Method for monitoring blood flow and metabolic uptake in tissue with radiolabeled alkanoic acid

ABSTRACT

The present invention relates to novel modified fatty acid analogs, where a positron or gamma-emitting label is placed at a position on a fatty acid backbone and an organic substituent is substituted at the 2,3; 3,4; 4,5; 5,6 and other sequence positions of a fatty acid backbone. These novel fatty acid analogs are designed to enter the tissues of interest by the same long chain fatty acid carrier mechanism as natural fatty acids, however, functional substituents in the 2,3; 3,4; 4,5; 5,6 and other sequence positions, block the catabolic pathway, thus trapping these analogs in a virtually unmodified form in the tissues of interest.

REFERENCE TO RELATED APPLICATIONS/INCORPORATION BY REFERENCE

This application claims priority to U.S. Application Ser. No.60/463,574, filed on Apr. 17, 2003.

Each of the applications and patents cited in this text, as well as eachdocument or reference cited in each of the applications and patents(including during the prosecution of each issued patent; “applicationcited documents”), and each of the U.S. and foreign applications orpatents corresponding to and/or claiming priority from any of theseapplications and patents, and each of the documents cited or referencedin each of the application cited documents, are hereby expresslyincorporated herein by reference. More generally, documents orreferences are cited in this text, either in a Reference List before theclaims, or in the text itself; and, each of these documents orreferences (“herein-cited references”), as well as each document orreference cited in each of the herein-cited references (including anymanufacturer's specifications, instructions, etc.), is hereby expresslyincorporated herein by reference. Documents incorporated by referenceinto this text may be employed in the practice of the invention. Thisapplication makes reference to U.S. Pat. No. 4,524,059, issued on Jun.18, 1985, the contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to novel radiolabeled fatty acid analogshaving a cyclic organic substituent, where a positron or gamma-emittinglabel is placed at a position on a fatty acid backbone and an organicsubstituent is substituted at the 2,3; 3,4; 4,5; 5,6 and other sequencepositions of a fatty acid backbone. These novel fatty acid analogs aredesigned to enter the tissues of interest by the same long chain fattyacid carrier mechanism as natural fatty acids, however, functionalsubstituents in the 2,3; 3,4; 4,5; 5,6 and other sequence positions,block the catabolic pathway, thus trapping these analogs in a virtuallyunmodified form in the tissues of interest.

BACKGROUND OF THE INVENTION

Many imaging modalities have been developed for non-invasive evaluationof heart disease. Radiotracers that can assess myocardial perfusion andheart metabolism are useful for clinical evaluation of ischemic heartdisease and cardiomyopathies. Fatty acids (FAs) are the principalsubstrate for the production of adenosine triphosphate (ATP) in themyocardium under aerobic conditions. Nearly 70% of myocardial energyresults from metabolism of fat in the basal state, with the remainder ofthe myocardial energy requirements supplied by glucose (15%), lactateand pyruvate (12%) and amino acids (5%). Therefore, FAs and modifiedfatty acids (MFAs) have been proposed for imaging the heart.

Uptake of free FAs by the myocardium occurs at an extraction percentageof 40% to 60% of blood content, which is proportional to perfusion.Transported to the heart as nonesterified FAs, as triglycerides in verylow-density lipoprotein particles or in chylomicrons, or bound to serumalbumin, they pass along concentration gradients to the interstitium.Under these conditions, FAs supply as much as 70% of oxidativelymetabolized substrate. The extraction of free FAs by the myocyte isregulated by several variables including FA chain length, theavailability of other metabolic substrates, circulating levels ofhormones, cardiac workload, and the presence or absence of ischemia.

MFAs behave like native FAs up to the β-oxidation step in themitochondria, where it is sequestered for a long period of time (Livni,E. et al. 1990 Lipids 25: 238-40). Highly simplified, the fate of an FAmay be described as follows: FA passes from capillary blood into theinterstitial space. It may “back-diffuse” to the vascular space or passthrough the interstitial space, where it may become activated asacyl-coenzymeA (CoA). The activated FA can then be esterified to formtriglycerides, incorporated into phospholipids or carried into themitochondria and oxidized. The activation of FA to acyl-CoA requiresenergy and is believed to be essentially irreversible in vivo. Sinceacyl-CoA cannot escape through the cell membrane, it becomes trapped inthe cell. However, the formation of triglycerides is not irreversibleand these can be broken down into the constituent FA and glycerol,adding to the FA pool.

As described above, there is an intimate relationship between FAmetabolism and myocardial integrity. As a result of their high rate ofutilization, labeled FAs in conjunction with suitable detectiontechniques can provide a means of quantifying in vivo regionalmyocardial metabolism. Two approaches can be employed to quantify theutilization of substrates in vivo. The first involves the use ofradiolabeled, positron emitting physiologic substrates. This approachhas been used to assess glucose and FA metabolism in the heart withcarbon 11(¹¹C) palmitate (PA) in rabbit (Raichle, M. E. et al. (1978)Science 199: 986-987). It has been demonstrated that after briefintervals of ischemia, PA extraction fell markedly, even whenalterations in cardiac function were reversible (Klein M. S. et al(1979) Am. J. Physiol. 237: H51-H58; Goldstein R. A. et al (1980) J.Nucl. Med. 21: 342-348; Weiss, E. S. et al. (1969) Circulation 19:25-32; Fox, K. A. et al. (1975) Circ. Res. 57: 232-243; Hoffman, E. J.et al. (1977) J. Nucl. Med. 18: 57-61; Ter-Pogossian, M. M. et al.(1980) Circulation 61: 242-255; Weiss E. S. et al. (1977) Circulation55: 66-73; Schon, H. R. et al. (1982) Am. Heart J. 103: 532-547; Schon,H. R. et al. (1982) Am. Heart J. 103: 548-561; Schelbert, H. R. et al.(1983) Am. Heart J. 105: 492-504; Schelbert, H. R. et al (1983) Am.Heart J. 106: 736-50; Sobel, B. E. (1982) Am. Heart J. 103: 673-681;Schelbert, H. R. et al. (1983) Am. Heart J. 105: 522-526; Schelbert, H.R. (1985) Circulation 72: TV122-133; Sobel, B. E. (1985) Circulation72:IV22-30; Rosamond, T. L. et al (1987) J Nucl. Med. 28: 1322-1329;Grover-McKay, M. et al (1986) Circulation 74: 281-292; Schelbert, H. R.et al (1986) Am. Heart J. 111: 1055-1064; Jaffe, A. S. et al (1987) Int.J. Cardiol. 15: 77-89; Knabb, R. M. et al (1987) J. Nucl. Med.28:1563-1570; Myars, D. W. et al (1987) Am. J. Physiol. 253: 107-114).In addition, zones of persistently decreased flow also showed decreasedPA extraction (Schelbert, H. R. (1985) Circulation 72: TV122-133).

Other studies using ¹¹C-PA provide the experimental basis for studyingregional myocardial FA distribution. The application of physiologicradiolabeled FAs to in vivo quantification of regional myocardialmetabolic rates suffers from several drawbacks. First, the use of¹¹C-FAs labeled with ¹¹C on the carboxyl group is subject to loss oflabeling during the first round of β-oxidation. Studies employing directintracoronary administration of ¹¹CO₂ and direct myocardial monitoringdemonstrate the evolution of ¹¹CO₂ within 30 seconds and a 50% clearancewithin 2-8 minutes. Secondly, the rapid washout of the radiolabel due toβ-oxidation and short sequential imaging periods imposes limitations oncounting statistics. Third, in the myocardial cell, FAs are distributedamong different pools: free FAs, triglycerides, phospholipids,diglycerides, and monoglycerides. It is unclear whether the rate of FAoxidation is proportional to the rate of triglyceride hydrolysis. Afterthe initial clearance of radioactivity from the blood, there is a risein activity due to the release of radiolabeled metabolites from theliver. This makes the completion of labeled FA detection necessarybefore the rise occurs. Other studies in dogs using ¹¹C-PA showed thatduring ischemia, quantitation is limited due to the complexity of itsmetabolic fate (Schelbert et al., 1983 Am. Heart J 106:736-50). ¹¹Cacetate has been proposed as an alternative due to its simpler metabolicfate.

The second approach involves the use of “analog tracers” that enter aknown metabolic pathway. However, because of their unique chemicalstructure, metabolism of these tracers stop at a certain stage, leavingthe radiolabel trapped in the cell in a known form. This concept hasbeen applied to the study of glucose metabolism using glucose analogssuch as 1-[¹¹C]-2-deoxyglucose (2 DG) and 2-¹⁸F]-fluorodeoxyglucose (2FDG). The principle of metabolic trapping has been used successfullywith 2 FDG to measure in vivo regional glucose metabolic rates inhumans. Investigations of the use of 2 FDG for measuring myocardialglucose metabolism have been conducted. Similarly, FAs have also beenwidely used to measure metabolic activity in the myocardium. A majordrawback to the use of FAs is the quick washout rates, as alluded above.FAs tend to wash out very quickly due to β-oxidation, depending on theposition of the radionuclide. Subsequently, the radiolabeled FA ormetabolites can then accumulate in tissues other than the region ofinterest, primarily liver and lung. In radiohalogenated aliphatic fattyacids, such accumulation occurs frequently with ¹²³I, which migrates andis stored in the thyroid gland, and ¹⁸F, which is stored in bone.

Evans and coworkers radiolabeled straight-chain FAs by saturation of thedouble carbon bond of oleic acid with ¹³¹I and found that althoughphotoscans of the canine heart were produced, the low specific activityof the final product precluded its clinical use (Evans, J. et al (1965)Circ. Res. 16: 1-10; reviewed in Corbett, J. R., (1999) Semin. Nucl.Med. 29(3): 1999-2006). Since then, there have been many MFAs developedfor cardiac imaging. Poe and coworkers showed that [¹²³I]-hexadecanoicacid (IHXA) and [¹²³I]-heptadecanoic acid (IHDA) were indicators ofmyocardial perfusion in experimental canine models and demonstratedclearance rates similar to that of ¹¹C-PA (Poe, N. et al, (1976) J.Nucl. Med. 16; 17-21; Poe, N. et al, (1977) Radiology 124: 419-424). Allsubjects with prior myocardial infarcts showed decreased regional traceruptake. Machulla et al reported that the c-terminal labeled FAs weremore efficiently extracted than analogs labeled in the cc-position, andthat IHDA had the highest uptake (Machulla, H. et al (1978) J. Nucl.Med. 19: 298-302).

Although IHDA and IHXA have potential as myocardial perfusion agents,their ability to access myocardial metabolism in patients has beenquestioned (Freundlieb, C. et al, (1980 J Nucl. Med. 21(11): 1043-50;Visser, F. C. et al, (1985) Circulation 72(3): 565-71). The clinicalutility of radiolabeled iodoalkyl FAs appears limited by: 1) the rapidappearance of free radioiodine, requiring special correction proceduresto differentiate between myocardial and blood pool activity; 2) shortelimination half-lives, making them unattractive agents for singlephoton imaging; and 3) data suggesting that the elimination rate may notreflect β-oxidation but rather de-iodination and back-diffusion of thetracer across the membrane. Further, protocols and algorithms developedfor planar imaging are not applicable to single photon imaging (SPECT),effectively eliminating it as a potential imaging modality for themeasurement of metabolic parameters with these radiotracers. Imagingdifficulties associated with de-iodination of IHDA and IHXA ultimatelyresulted in the development of the branched FAs.

The molecular structure of FAs can be modified to attenuate myocardialmetabolism, prolong cardiac retention, and avoid washout effects. Toprevent rapid de-iodination of alkyl FAs and promote stabilization ofthe iodine radiolabel, 15-(p-iodophenyl)pentadecanoic acid (IPPA) wasdeveloped as an alternative (Machulla, H. et al (1980) Eur. J. Nucl.Med. 5: 171-173). The ¹²³I-label attached to a terminal phenyl ring ineither the ortho or para position is stabilized against de-iodination.[¹²³I]-IPPA has kinetics similar to the physiological substrate ¹¹C-PAin perfused rat hearts (Reske, S. et al, (1984) J. Nucl. Med. 25:1335-1342). The uptake of IPPA is related to perfusion, and IPPAgenerally follows the normal metabolic pathway for β-oxidation(Caldwell, J. et al, (1990) J. Nucl. Med. 31: 99-105). Iodobenzoic acidand its metabolite iodohippurate are the products of IPPA oxidation,which are rapidly excreted by the kidneys with the iodine moiety stillattached, preventing buildup of free radioiodine.

Studies using myocardial biopsy specimens have shown rapid extraction bynormal myocardium, with biexponential clearance, including a fastcomponent t_(1/2) of 3.5 minutes (flow), a slow component t_(1/2)=130minutes (metabolism), and a blood clearance t_(1/2)=5 minutes(elimination) (Chien, K. et al (1983) Am. J. Physiol. 245: H693-H697).Compared with alkyl straight-chain FAs, IPPA has the advantages of rapidmyocardial uptake, iodine stabilization, and rapid clearance ofmetabolites from the body. While IPPA was a significant improvement overthe straight chain FAs, providing excellent image quality, andpermitting SPECT image acquisition and quantification with estimates ofmetabolic rates, the rate of IPPA metabolism and clearance was stillrelatively fast for SPECT imaging. Thus, an effort was made to developradiolabeled FA analogs with attenuated oxidative metabolism.

Methyl branching was introduced to slow myocardial clearance and improvequantitative image accuracy (Livni, E. et al. 1982 J. Nucl. Med.23:169-75; Elmaleh, D. R. et al. 1981. J. Nucl. Med. 22: 994-9; Elmaleh, D.R. et al. 1983 Int. J. Nucl. Med. Biol. 10:181-7; Goodman, M. M. et al.1984 J. Org. Chem. 49: 2322-5; Livni, E. et al. 1985. Eur. Heart J. 6(Suppl B): 85-9; Bianco, J. A. et al. Eur. J. Nucl. Med. 12: 120-4). Theaddition of methyl group(s) at the 3-carbon position blocks β-oxidationby preventing formation of the 3-carboxy-intermediate (β-ketoacyl-ScoA)via the dehydrogenation of the 5-L-hydroxy ScoA intermediate. Twoiodine-labeled MFAs that provide prolonged myocardial retention are15-(p-iodophenyl)-3-(R,S)-methyl-pentadecanoic acid (BMIPP) and15-p-iodophenyl)-3,3-dimethyl-pentadecanoic acid (DMIPP). The kineticsand subcellular distribution of these methyl-branched FAs have beenevaluated, with DMIPP demonstrating the greatest myocardial retentiontimes, with no significant metabolism (Knapp, F. et al, (1986) Eur. J.Nucl. Med. 12: S39-S44; Ambrose, K. et al, (1987) Eur. J. Nucl. Med. 12:486-491). However, DMIPP demonstrates very slow myocardial clearance(6-7 hours), limiting its usefulness for certain applications.Additionally, DMIPP has been detected in exogenous tissues, such as theliver (Sloof et al, (1997) Nucl. Med. Commun. 18(11): 1065-70). BMIPPalso accumulates in the liver, but to a lesser extent than DMIPP.

BMIPP is currently the most widely used radiolabeled MFA for cardiacimaging. BMIPP has prolonged myocardial retention (30-45 minutes) andundergoes β-oxidation in the myocyte after the initial α-oxidation andoxidative decarboxylation, producing α-hydroxy-BMIPP as an intermediate(Yamamichi, Y. et al, (1995) J. Nucl. Med. 36: 1042-1050). After loss ofpropionic acid, further degradation proceeds through successive cyclesof β-oxidation to the end product, (p-iodophenyl)-acetic acid.Additionally, it has been shown that initial distribution of BMIPP inthe first several minutes after injection is comparable to that ofperfusion tracers like ²⁰¹Thallium and ^(99m)technetium compounds. Thus,it can be argued that the use of BMIPP alone imaged early and late afterinjection is all that is required to evaluate myocardial viability witha high degree of accuracy. Likewise, numerous groups have accomplishedwork on FAs as potential markers for blood flow and their collectivedata determined that a single injection of certain FAs produces imagesthat are similar to those produced by ²⁰¹Tl, ¹¹C-PA, or potassium (vander Wall, E. E. et al, (1980) Eur. J. Nucl. Med. 5(5): 401-5; Kairento,A. L. et al, (1988) Int. J. Rad. Appl. Instrum. B. 15(3): 333-8;Kobayashi, H. et al, (1997) J. Nucl. Med. 39(7): 1117-22; Kawamoto, M.et al, (1994) J. Nucl. Cardiol. 1(6): 522-8). Thus, a single injectionlimits the amount of radioactivity exposure to the patient, as well asbeing cost-effective. While BMIPP is a widely used FA for both flow andmetabolism, there is a need in the art for MFAs that have longermyocardial retention without appreciable metabolism and migration of theradiolabel to other unwanted areas. BMIPP and its metabolites have beendetected at significant levels in lung tissue (Sloof et al, (1997) Nucl.Med. Commun. 18(11): 1065-70).

While there are a plurality of MFAs that are suitable for imaging, manyeither undergo significant β-oxidation, resulting in production ofradiolabeled metabolites that can accumulate in other tissues, or failto be efficiently transported into the myocardium, resulting inback-diffusion and re-esterification into the triglyceride pool. Thus,there is a need in the art for a radiolabeled MFA analog that istransported to the tissue of interest by endogenous, physiologicalmeans, but fails to undergo β-oxidation due to the presence of branchedorganic substituents. This would allow the MFA to be retained in avirtually unmodified form for a sufficient amount of time in the tissueof interest (i.e. cardiac tissue) to be detected by conventional means.Further, there is a need in the art for a radiolabeled MFA that sharesthe aforementioned characteristics and is retained without significantmigration to unwanted areas of the organism.

SUMMARY OF THE INVENTION

Alterations in FA uptake are considered to be sensitive markers ofischemia and myocardial damage. Radiolabeled FAs that initially displaymyocardial blood flow and eventually show adequate myocardial retentionassociated with metabolic activity are attractive candidates forclinical evaluation of regional discrepancies in heart blood flow and FAmetabolism, which occur in ischemic heart disease and cardiomyopathies.

Therefore, the present invention relates to novel radiolabeled FAanalogs having a cyclic organic substituent, where a positron orgamma-emitting label is placed at a position on an FA backbone and anorganic substituent is substituted at the 2,3; 3,4; 4,5; 5,6 and othersequence positions of an FA backbone. These novel FA analogs aredesigned to enter the tissues of interest by the same long chain FAcarrier mechanism as natural FAs, however, functional substituents inthe 2,3; 3,4; 4,5; 5,6 and other sequence positions, block the catabolicpathway, thus trapping these analogs in the tissues of interest.

Accordingly, in one aspect of the present invention, a radioactivelylabeled analog of a fatty acid that is taken up by mammalian tissue isprovided, comprising the formula:R—(CH₂)_(n)-Z-(CH₂)_(m)CO₂H,wherein n is 8-22, m is 0-10, R is a CH₃, aryl or a heterocyclic group,and Z is a cyclic or heterocyclic organic substituent which causes saidanalog to be metabolically trapped in said tissue.

The term “aryl” in the context of this application may comprise, but isnot limited to, a 5-, 6- or 7-membered ring. Further, the term “cyclic”can refer to cyclic alkanes such as cyclopropyl, cyclobutyl, andcyclopentyl, but is not so limited. Similarly, “heterocylic” can referto any 3 to 5-membered ring structure that can comprise, for example,nitrogen, sulfur, or oxygen atoms.

In another aspect, a radioactively labeled analog of a fatty acid thatis taken up by mammalian tissue is provided, comprising the formula:

wherein D is CH₂ or CH₂CH₂, E is CH₂ or CH₂CH₂, m is 0-10, n is 8-14 andR is a CH₃, aryl or a heterocyclic group, wherein cyclic organicsubstituent —CDCE- causes said analog to be metabolically trapped insaid tissue.

Another aspect of the present invention provides a radioactively labeledanalog of a fatty acid, comprising the formula:

wherein A can be (CH₂)_(x), O, or S; X is 1, 2, 3, and 4; cis and transisomers R,R and S,S and their racemic forms; m is 0-10; n is 14-8; R is¹⁸F-phenyl, or ¹²³I-phenyl, and wherein the cyclic or heterocyclicorganic substituent —CH-A-CH— causes said analog to be metabolicallytrapped in said tissue.

In another aspect of the present invention, a radioactively labeledanalog of a fatty acid that is taken up by mammalian tissue is provided,comprising the formula:

wherein A is (CH₂)_(y), O, or S; y is 1, 2, 3, 4; cis and trans isomersR,R, and S,S and their racemic forms; m is 0-10; n is 14-8; p is 0-6; Ris CH₃; and X is a radioactive label.

The present invention also provides a radioactively labeled analog of afatty acid that is taken up by mammalian tissue, comprising the formula:

wherein X can be H, ¹⁸F, ¹²³I, ¹³¹I, ^(34m)Cl, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, alkyland heteroalkyls thereof; Y can be H, ¹⁸F, ¹²³I, ¹³¹I, ^(34m)Cl, ⁷⁵Br,⁷⁶Br, ⁷⁷Br, alkyl and heteroalkyls thereof; A is (CH₂)_(z), O or S; Z is1-4, m is 0-10, n is 8-14, p is 0-6, R is CH₃, aryl or heterocyclic, andwherein the cyclic or heterocyclic organic substituent —CH-A-CH— causessaid analog to be metabolically trapped in said tissue.

Another aspect of the present invention provides a radioactively labeledanalog of a fatty acid that is taken up by mammalian tissue, comprisingthe formula:

wherein A is (CH₂)_(z), O or S, y is 1-4, m is 1-10, n is 8-14, p is0-6, R is CH₃, aryl or heterocyclic, X can be ¹⁸F, ¹²³I, ¹³¹, ^(34m)Cl,⁷⁵Br, ⁷⁶Br or ⁷⁷Br; and wherein the cyclic or heterocyclic organicsubstituent —CH-A-CH— causes said analog to be metabolically trapped insaid tissue.

Preferably, the novel fatty acid has a cyclic organic substituentlabeled with ¹⁸F, ¹²³I, ¹³¹I, ^(34m)Cl, ⁷⁵Br, ⁷⁶Br and ⁷⁷Br.

In another embodiment, the novel fatty acid having a cyclic organicsubstituent is saturated.

Another embodiment of the present invention describes the novel fattyacid having a cyclic organic substituent that contains one or moredouble bonds.

In one embodiment, the organic substituent is a 3-to-5 membered cyclicstructure.

Another aspect of the present invention provides a method of measuringblood flow in a subject, comprising the following steps:

-   -   a) localizing a detectable amount of a FA composition of the        invention to a tissue of interest;    -   b) detecting a signal from said FA composition in a tissue of        interest within about 1 minutes and about 5 minutes after        administration;    -   c) imaging a tissue of interest and;    -   d) determining the rate of blood flow in a tissue of interest.

In another aspect, a method for measuring metabolism in a subjectcomprises the following steps:

-   -   a) localizing a detectable amount of a FA composition of the        invention to a tissue of interest;    -   b) detecting a signal from said FA composition in a tissue of        interest within about 30 minutes and about 120 minutes after        administration;    -   c) imaging a tissue of interest and;    -   d) determining the rate of metabolism in a tissue of interest.

Another aspect of the present invention provides a method for retaininga fatty acid composition of the invention in a tissue of interest,comprising the steps of:

-   -   a. localizing a detectable amount of the composition to the        tissue;    -   b. retaining the composition, or a metabolic derivative thereof,        in the tissue by reducing transport and back-diffusion of the        composition; and    -   c. detecting the composition or the metabolic derivative in the        tissue.

The present invention further provides a method for retaining a fattyacid composition of the invention in a tissue of interest, comprisingthe steps of:

-   -   a. localizing a detectable amount of the composition to the        tissue;    -   b. retaining the composition, or a metabolic derivative thereof,        in the tissue by reducing dehydrogenation of the composition;        and    -   c. detecting the composition or the metabolic derivative in the        tissue.

Another aspect provides a method for retaining a fatty acid compositionof the invention in a tissue of interest, comprising the steps of:

-   -   a. localizing a detectable amount of the composition to the        tissue;    -   b. retaining the composition, or a metabolic derivative thereof,        in the tissue by reducing hydroxylation of the composition; and    -   c. detecting the composition or the metabolic derivative in the        tissue.

The present invention further provides a method for retaining a fattyacid composition of the invention in a tissue of interest, comprisingthe steps of:

-   -   a. localizing a detectable amount of the composition to the        tissue;    -   b. retaining the composition, or a metabolic derivative thereof,        in the tissue by reducing ketoacyl formation of the composition;        and    -   c. detecting the composition or the metabolic derivative in the        tissue.

Yet another aspect of the present invention provides a method forretaining a fatty acid composition of the invention in a tissue ofinterest, comprising the steps of:

-   -   a. localizing a detectable amount of the composition to the        tissue;    -   b. retaining the composition, or a metabolic derivative thereof,        in the tissue by reducing ketoacetyl elimination of the        composition; and    -   c. detecting the composition or the metabolic derivative in the        tissue.

In one embodiment, the tissue of interest is heart tissue.

In another embodiment, the tissue of interest is liver tissue.

The present invention preferably describes tumor tissue as the tissue ofinterest.

In one embodiment, the tissue is diseased.

In another embodiment, the tissue is healthy.

Preferably, the tissue can be subjected to exercise-induced stress orpharmacologically induced stress.

Another aspect of the present invention provides a method ofsynthesizing a fatty acid composition of the invention, comprising thesteps of:

-   -   a) synthesizing a mono-protected primary alcohol from a starting        compound;    -   b) adding a cyclic or heterocyclic organic substituent to the        mono-protected primary alcohol to form a cyclic mono-protected        primary alcohol; and    -   c) treating the cyclic mono-protected primary alcohol to form        the fatty acid analog.

One embodiment of the present invention describes the starting compoundcomprising a carbon backbone that is saturated.

In another embodiment, the starting compound comprises a carbon backbonethat unsaturated.

Another embodiment describes the starting compound comprising a terminalphenyl group.

In another embodiment, the starting compound is a cyclic primaryalcohol.

One embodiment of the present invention describes a cyclic alkane as thecyclic organic substituent.

Another embodiment describes the heterocyclic organic substituent as a3-5-membered heterocyclic ring structure.

In another embodiment, the method further comprises adding a radioactivelabel that is bonded to a carbon atom of the analog.

The present invention further provides a kit for administration of aradioactively labeled analog of a fatty acid, comprising an analog of afatty acid synthesized according to the methods of the invention, aradioactive isotope, a pharmaceutically acceptable carrier, andoptionally instructions for preparing the radioactively labeled analogor use thereof.

Preferably, the radioactive isotope is selected from the groupconsisting of ¹⁸F, ¹²³I, ¹³I, ^(34m)Cl, ⁷⁵Br, ⁷⁶ Br and ⁷⁷Br.

Throughout this specification and claims, the word “comprise” orvariations such as “comprises” or “comprising” will be understood toimply the inclusion of a stated integer or group of integers but not theexclusion of any other integer or group of integers.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following Detailed Description, given by way of example, but notintended to limit the invention to specific embodiments described, maybe understood in conjunction with the accompanying Figures, incorporatedherein by reference.

FIG. 1 is a schematic overview of the synthesis of[¹⁸F]-9-fluoro-3,4-cyclopropyl-heptadecanoic acid.

FIG. 2 depicts the general formula of a saturated fatty acid comprisinga substituted radiolabel directly on the fatty acid backbone. Thisfigure of a fatty acid variant comprises a generalized structure similarto the compound in FIG. 1.

FIG. 3 depicts [¹⁸F]-FCPHA activity in the heart of a monkey as afunction of time.

FIG. 4 shows heart images of a pig at 2-8 minutes after intravenousinjection of 18 mCi of [¹³N]-ammonia.

FIG. 5 shows heart images of the same pig in FIG. 5, at 2-8 minutesafter intravenous injection of 19 mCi [¹⁸F]-FCPHA.

FIG. 6 is a comparison of [¹³N]-ammonia (right) and [¹⁸F]FCPHA (left)images obtained from a pig at 2-8 minutes after tracer injection.

FIG. 7 is an image collected by positron emission tomography of thebiodistribution of [¹⁸F]-FCPHA in the left and right ventricles of amonkey heart.

FIG. 8 shows transverse heart level slices of a monkey 60 minutes afteradministration of [¹⁸F]-FCPHA.

FIG. 9 depicts the general formula of a terminally labeled straightchain fatty acid comprising a substituent designated by ‘A’ and a phenylmoiety comprising a substituted radiolabel.

FIG. 10 is a schematic overview of the synthesis of endo-[¹⁸F]fluoro- or[¹²³I] iodo-3,4-cyclopropylheptadecanoic acid.

FIG. 11 depicts the general formula of a fatty acid comprising anendo-vinyl group. This endo-vinyl group can comprise a substitutedradiolabel at substituents ‘X’ or ‘Y’.

FIG. 12 is a schematic overview of the synthesis of exo-[¹⁸F]fluoro or[¹²³I] iodo-3,4-cyclopropylheptadecanoic acid.

FIG. 13 depicts the general formula of a fatty acid comprising anexo-vinyl group. This exo-vinyl group can comprise a substitutedradiolabel at substituents ‘X’ or ‘Y’.

FIG. 14 shows the partial synthesis of a portion of a modified fattyacid comprising a substituent that can be a four- to five-membered ringstructure.

FIG. 15 shows the general formula of a modified fatty acid comprising asubstituent that can be a four- to five-membered ring structure. Thisfatty acid variant can comprise a combination of features described inFIGS. 2, 4, 11, and 13.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the instant invention, novel radiolabeled fatty acidcompositions having a cyclic organic substituent can be administered toan animal, including a human, to determine both blood flow to the organand metabolism by an organ of the body of the animal. In the presentapplication, the term “modified fatty acid” can be regarded as asynthetic or naturally occurring fatty acid that has been syntheticallymodified. Also within the context of this application, the term “organicsubstituent” refers to organic chemical structures bonded to the fattyacid that is effective in decreasing the in vivo rate of β-oxidation ofthe fatty acid in tissues of interest.

The novel FAs described herein are radiolabeled and are modified with anorganic substituent at 2,3; 3,4; 4,5; 5,6 and other sequence positions.The term “2,3” refers to the carbon bond between the carbon atomslocated at position C2 and C3 (counting from the carboxyl carbon atom).As such, the related term “3,4” refers to the carbon atoms at positionsC3 and C4. Likewise, the term “2,3” could also be interpreted by theskilled artisan to correspond to the term “beta-gamma” in reference tothe carbon atoms, or “βγ”. The related term “3,4” therefore, could beinterpreted to correspond to the term “gamma-delta” in reference to thecarbon atoms, or “γδ”. The carboxyl end of the molecule will hereinafterbe referred to as the “right” end of the molecule, while the oppositeend will be referred to as the “left” end of the molecule. Compounds canhave the general formula, also depicted in FIGS. 2 and 4:

In all of the preceding embodiments, the organic substituent may besaturated or unsaturated. The organic substituent may also comprise atleast one heteroatom, advantageously N, O or S, most advantageously O orS. Nitrogen can also be used and it is well within the skill of theartisan to determine proper substitution of the nitrogen atom, as wellas determining the appropriate biodistribution of the resultant analog.The carbon backbone of the fatty acid may also be substituted with atleast one heteroatom herein defined.

It is further understood that A can be a C₁-C₆ alkyl, alkenyl oralkynyl, wherein one or more of the C atoms, advantageously 1, 2 or 3 Catoms, are substituted by a heteroatom, advantageously N, O or S, mostadvantageously O or S. It is well within the scope of one skilled in theart to determine proper placement of the nitrogen atom, in addition todetermining the appropriate biodistribution of the resultant analog.

Further, “aryl” as herein defined may comprise, but is not limited to, a5-, 6- or 7-membered ring. Further, “heteroatom” as herein defined maycomprise, but is not limited to, 1, 2 or 3 heteroatoms.

The chain length of the FA also affects the tissue by which it isprimarily taken up. Generally, a chain length of 12-20 carbon atoms,inclusive, is optimal for selective uptake by myocardial tissue, whilethe liver will selectively take up a chain length of 5-11 carbon atoms,inclusive. The carbon chain of the analog can be saturated orunsaturated. A preferred embodiment of the invention is a FA backbonethat is saturated and has a cyclic organic substituent.

In another embodiment of the present invention, the FA backbone containsone or more vinyl groups, resulting in unsaturation of one or morecarbon-carbon bonds. Preferably, the vinyl group or groups are placed onthe opposite side of the carboxyl group, or to the left of thesubstituent designated by the letter ‘A’ in the general formula shownabove. In another preferred embodiment, the vinyl groups are appended tothe FA backbone such that the vinylic substituent is branched from theFA chain. More preferably, 1-6 carbon atoms separate the vinyl group(s)from the substituent designated by ‘A’. Generalized drawings of thesepreferred embodiments are provided in FIGS. 6 and 8.

Activation of FAs is an energy-dependent step necessary for theirtransport into and sequestration within tissues of interest. Preferredembodiments of the instant invention are related to manipulation of theFA metabolism pathway, also referred to herein as “β-oxidation”. Thisprocess begins with acyl-coenzyme A (CoA) synthase, which activatescytosolic free FAs by decarboxylation of the terminal COOH at the outermitochondrial membrane in the presence of adenosine triphosphate (ATP).This forms an acyl-adenylate mixed anhydride, which reacts with CoA toform fatty acyl-CoA and AMP (adenosine monophosphate). One skilled inthe art will know that different isoforms of acyl-CoA synthase arespecific for FAs of varying chain lengths. In the context of thisapplication, the term “CoA” is used synonymously with “ScoA”.

Failure to activate FAs to acyl-CoA often results in “back-diffusion”into the vascular space and as a result, these back-diffused FAs are notmetabolized. However, once activated, FAs can be “transported” acrossthe inner mitochondrial membranes and undergo stepwise β-oxidation.Transport across the inner mitochondrial membrane is also referred to inthe context of this application as “metabolic trapping” or “metabolicretention” of FAs. Activated fatty acyl-CoA cannot be directlytransported across the inner mitochondrial membranes and the acyl chainmust be transferred to carnitine by an acyl-carnitine/carnitinetransporter. This facilitated diffusion through the inner mitochondrialmembrane is the rate-limiting step for the oxidation of FAs. During highcardiac workloads or during myocardial ischemia, esterified FAs arediverted into storage pools as cytosolic triglycerides and membranephospholipids. It is contemplated that activation of the novel analogsdescribed by the invention and transport into the metabolizing tissue ofinterest occurs normally, but subsequent steps relating to disassemblyof the analog are blocked.

Inside the mitochondrial matrix, FAs are metabolized in four steps. 1)Formation of a trans-2,3-double bond occurs through acyl-CoAdehydrogenase from a fatty acyl-CoA precursor to formtrans-Δ²-enoyl-CoA. 2) The trans double bond is then hydrated byenoyl-CoA hydratase to form 3-L-hydroxyacyl-CoA, which is subsequently3) dehydrogenated by 3-L-hydroxylacyl-CoA dehydrogenase to formβ-ketoacyl-CoA. 4) Cleavage of the 2,3 carbon-carbon bond is facilitatedthrough β-ketoacyl-CoA thiolase to form acetyl-CoA and a new acyl-CoAthat is two carbons shorter than its starting molecule. This process isrepeated for each remaining two-carbon fragment.

The present invention relates to FAs having a cyclic organic substituentthat cause attenuation of the β-oxidation pathway by potentiallypreventing or blocking the metabolic sequence of one or several ways.For example, the intermediate is not a substrate for one of the enzymesrequired during the β-oxidation pathway or the intermediate cannotundergo a metabolic hydrogenation, dehydrogenation or hydroxylationstep. An early trapping step that is related to flow and initial uptakemay be advantageous for acquiring blood flow related images. Metabolictrapping or retention that occurs after one or several metabolic stepscan represent the metabolic integrity of the target tissue. “Reducing”the formation of metabolic derivatives of the FAs of the invention canmean total or partial prevention or elimination of metabolic reactionssuch as, but not limited to, transport and back-diffusion,hydrogenation, dehydrogenation, hydroxylation, ketoacyl formation, andketoacetyl elimination. As such, one aspect of the present inventionreduces formation of β-ketoacyl-CoA (step 3). Yet another aspect reducesformation of a trans-Δ²-enoyl-CoA derivative (step 1). Still anotheraspect of the invention reduces formation of the products of subsequentβ-oxidation steps, depending on the organic substituents and theirplacement on the FA backbone. The position of the organic substituent onthe FA backbone can determine the extent of which the molecule undergoesβ-oxidation and metabolism.

Modifications of the FA, such as the presence of unsaturated cis-doublebonds, can also cause attenuation of FA metabolism. A cis-3,4-doublebond is not a substrate for enoyl-CoA hydratase and requires enoyl-CoAisomerase, which mediates conversion of the cis double bond to the morestable, ester-conjugated trans form. Once this isomerization eventoccurs, metabolism occurs normally until the fifth round of β-oxidation,where the presence of another double bond at an even-numbered carbonatom results in formation of 2,4-dienoyl-CoA. This molecule is a poorsubstrate for enoyl-CoA hydratase and requires another enzyme,NADPH-dependent 2,4-dienoyl-CoA reductase. The resultant molecule,trans-2-enoyl-CoA, is further isomerized to yield trans-3-enoyl-CoA by3,2-enoyl-CoA isomerase. Likewise, addition of other organicsubstituents, such as branched methyl or phenyl groups, take advantageof β-oxidation pathway attenuation, which can result in longer retentiontimes in the metabolizing tissue of interest.

An embodiment of the present invention is substitution of an organicsubstituent or substituents on the FA chain backbone. This organicsubstituent can be placed at 2,3; 3,4; 4,5; 5,6 and other sequencepositions, yielding the addition of the substituent at positions thatare branched from the FA backbone. The position of the organicsubstituent on the FA backbone may determine the extent of which themolecule undergoes β-oxidation and metabolism. In a preferredembodiment, an organic substituent bonded at the 3,4 position causes theanalog to be metabolically trapped in tissues of interest bysubstitution of CoA for the carboxyl carbon atom of the FA analog. Thenext two steps of metabolism of the resulting fatty acyl-CoA molecule,formation of trans-Δ²-enoyl-CoA and 3-L-hydroxyacyl-CoA derivatives,occur normally with 3,4-substitution. However, the resulting3-L-hydroxyacyl-CoA derivative is not a substrate for3-L-hydroxyacyl-CoA dehydrogenase due to the presence of the organicsubstituent and thus, β-ketoacyl CoA formation is prevented. In anotherpreferred embodiment, an organic substituent bonded at the 2,3 positioncauses the analog to be metabolically trapped in tissues of interest bysubstitution of CoA for the carboxyl carbon atom of the FA analog.However, the first metabolic step, which involves dehydrogenation of theanalog to ostensibly form a trans-Δ²-enoyl-CoA derivative, does notoccur, thus preventing any further metabolism of the analog. It iscontemplated that these specific embodiments can be used advantageouslyto measure blood flow.

Similarly, if an organic substituent is in position 4,5 and in othersubsequent positions on the FA backbone (i.e. 5,6; 6,7; etc), one ormore steps in β-oxidation may be completed and then be subsequentlyblocked. Yet another preferred embodiment of the invention contemplatesappending an organic substituent at positions further on the FA backbonefrom the carboxyl-terminal end, such as but not limited to the 4,5; 5,6;6,7 positions and so on. It will be apparent to those skilled in the artthat the position of the organic substituent on the FA backbone willdetermine the extent of which the molecule undergoes β-oxidation. Inthis embodiment, metabolic activity of the instant invention can beadvantageously measured due to its progression through the β-oxidationpathway.

Any organic substituent of the analog should be small enough to permitthe formation of the first chemical intermediate involved in the fattyacid β-oxidation process; too large a substituent can alter the uptakeand behavior of an analog to an undesirable extent. The chemical nature,as well as the size, of any substituent can affect the properties of theanalog. Generally, an analog having a substituent which does not renderthe analog excessively polar, e.g., an unsubstituted alkyl group, istaken up primarily by the heart, while an analog containing a polarizinggroup, e.g., an alcohol or an ether, will be taken up primarily by theliver.

In yet another aspect of the present invention, the functional groupdesignated by ‘A’ in the general formula provided above can comprisebranched alkyl groups, dimethyl groups, cyclic alkanes such ascyclopropyl, cyclobutyl, cyclopentyl, or any 3 to 5-memberedheterocyclic ring structure. The heterocyclic rings can containnitrogen, sulfur or oxygen atoms. A preferred embodiment of the instantinvention is a cyclopropyl ring substituent. Other preferred embodimentsof the invention contemplate cyclobutyl and cyclopentyl ringsubstituents, such as those described in the general diagram of FIG. 10.

Yet another object of the present invention contemplates that theorganic substituent may be appended onto the FA backbone in either cisor trans form. A method of cyclopropanation (Simmons-Smith reaction) isdescribed in the Examples section of this application and the improvedprocedure originates from Charette et al, J. Org. Chem. (1995) 60: 1081.The components of the reaction can be present as cis or trans isomer,depending on the FA precursor used for the addition of the cyclopropylmoiety. The cis diastereomer is preferentially synthesized by using 3equivalents of the pre-formed complex of Zn(CH₂I)₂.DME in CH₂Cl₂ (seeExample 1 for details on this complex) and incubation for 3 hours atbelow −10° C., whereas the cis isomer is made by using the cis olefinand the pre-formed complex and incubating the mixture for 2 hours atbelow −10° C. One skilled in the art will understand that thecyclopropyl group on the FA backbone has two chiral centers and thus themolecule exists in two enantiomeric forms: R, R or S, S. An embodimentof the present invention encompasses each diastereomer and theirrespective enantiomers, as well as any racemic mixtures or mesocompounds thereof.

The novel fatty acids according to the present invention can beradiolabeled with a positron or gamma-emitting label that is well knownin the art. Preferred embodiments of the invention comprise ¹⁸F, ¹²³I,¹³¹I, ^(34m)Cl, ⁷⁵Br, ⁷⁶Br or ⁷⁷Br. One of ordinary skill in the artwill recognize that different radionuclides display differentpharmacokinetic properties, such as elimination, clearance from and/oraccumulation in biological tissues, and half-life (t_(1/2)).Radionuclides are typically synthesized by a cyclotron, whichaccelerates subatomic particles from an ion source along a circularorbit. Particle acceleration inside a chamber is controlled by twoalternating electromagnetic fields. These accelerated particles can gainenergy and collide with a target at close to the speed of light.Bombardment of particles against the target result in unstable,radioactive isotopes, which are then attached to biologically relevantmolecules such as those exemplified by the instant invention.Alternatively, commercially available radionuclides are widely used andmay be appended onto biologically relevant molecules by chemicalsynthesis techniques well known in the art. Typically, the half-lives ofradiotracers used in imaging are relatively short, and thus manycyclotrons are key features of radiotracer detection apparatuses, suchas PET and SPECT scanners, or gamma cameras. One skilled in the art willunderstand the principles of radioisotopic decay and this concept willnot be discussed further in this application.

A preferred embodiment of the present invention is addition of apositron or gamma-emitting radiolabel at a position on the FA backbonethat prevents significant loss of the radiolabel during FA metabolismand migration to other tissues. One of sufficient skill in the art willappreciate that radiolabeling at the terminal carboxyl group is notrecommended, since this carboxyl group is removed by specific enzymesduring the early metabolic stages of the FA into the tissues ofinterest. In one aspect of the invention, the radiolabel is added to the9-carbon position of the FA backbone. In another aspect of theinvention, the radiolabel is added to a terminal phenyl group on the FAbackbone located on the opposing side from the carboxylic acid group. Inyet another aspect of the invention, one or more carbon-carbon bonds onthe FA backbone is unsaturated, resulting in a vinyl group and theradiolabel is appended directly to the vinyl group. In still anotheraspect of the invention, a radiolabeled vinyl group is appended onto theFA backbone and is branched from the FA backbone.

The radiolabeled FAs having a cyclic organic substituent can besynthesized by traditional organic chemical syntheses well known in theart. Similarly, the instant invention may be purified and analyzed by avariety of methods, including column purification, thin-layerchromatography (TLC), reverse-phase chromatography, high-performanceliquid chromatography (HPLC), gas chromatography (GC), infraredspectroscopy (IR), nuclear magnetic resonance (NMR) including variationssuch as correlation spectroscopy (COSY), nuclear Overhauser effectspectroscopy (NOESY), and rotating frame nuclear Overhauser effectspectroscopy (ROESY), and Fourier Transform, other analytical techniquessuch as mass spectrometry (MS) and variations thereof, includingelectrospray, chemical ionization, matrix assisted laser desorptionionization, time-of-flight, fast atom bombardment/liquid secondaryionization, among many other techniques.

The pharmaceutical compositions comprising the present invention, novelradiolabeled FAs that have cyclic organic substituents, may be in avariety of conventional depot forms. These include, for example, solid,semi-solid and liquid dosage forms, such as tablets, pills, powders,liquid solutions or suspensions, liposomes, capsules, suppositories,injectable and infusible solutions. Such dosage forms may includepharmaceutically acceptable carriers and adjuvants, which are well knownto those skilled in the art. These carriers and adjuvants include, forexample, RIBI, ISCOM, ion exchangers, alumina, aluminum stearate,lecithin, serum proteins, such as human serum albumin, buffersubstances, such as phosphates, glycine, sorbic acid, potassium sorbate,partial glyceride mixtures of saturated vegetable fatty acids, water,salts or electrolytes such as protamine sulfate, disodium hydrogenphosphate, sodium chloride, zinc salts, colloidal silica, magnesiumtrisilicate, polyvinyl pyrrolidone, cellulose-based substances, andpolyethylene glycol. Adjuvants for topical or gel base forms may beselected from the group consisting of sodium carboxymethylcellulose,polyacrylates, polyoxyethylene-polyoxypropylene-block polymers,polyethylene glycol, and wood wax alcohols. The preferred form of theinstant invention is an injectable form. Thus, this form may be subjectto other treatments during preparation to improve their tolerance insubjects.

As stated above, the animal of interest, preferably a human can beinjected with the radiolabeled FAs having a cyclic organic substituent.Any pharmaceutically acceptable dosage route, including parenteral,intravenous, intramuscular, intralesional or subcutaneous injection, maybe used to administer the novel FA compositions of the instantinvention. For example, the composition may be administered to thesubject in any pharmaceutically acceptable dosage form including thosewhich may be administered to a patient intravenously as bolus or bycontinued infusion over a period of hours, days, weeks, or months,intramuscularly—including paravertebrally andperiarticularly—subcutaneously, intracutaneously, intra-articularly,intrasynovially, intrathecally, intralesionally, periostally, or by oralor topical routes. Preferably, the compositions of the invention are inthe form of a unit dose and will usually be administered intravenouslyby bolus injection.

The purified FA can be contained in solutions such as saline, atconcentrations suitable for intravenous delivery. These FAs can becomplexed with albumin, a serum protein that binds to non-esterified FAsin the bloodstream. Typically albumin is added at a concentration of2-10%, more preferably between 4-6%. Alternatively, these FAs can beemulsified in non-ionic detergents such as but not limited to,polyoxyethylene sorbitan monolaurate derivatives (Tween), Nonidet P-40,β-D-octylglucoside, ursodeoxycholic acid (UDCA), or Triton X-100, andresuspended in solutions containing or lacking albumin prior toinjection. The dosages of radiolabeled cyclic substituted FA must bedetermined for each subject prior to administration, and the typicaldosage ranges can be between 0.1-25 mCi, more preferably between 1-5mCi.

In yet another aspect, the pharmaceutical compositions comprising theFAs according to the present invention may also be administered to anyanimal, including, but not limited to, horses, cattle, monkeys, birds,pet animals, such as dogs, cats, birds, ferrets, hamsters, rodents,squirrels, birds, and rabbits. The preferred embodiment of the inventionis to monitor diseases or disease states associated with blood flow, FAmetabolism or for imaging an organ of interest in a human.

The present invention further comprises methods to measure and/oridentify changes in blood flow and metabolism in tissues of interest inresponse to disease states, exercise, pharmacological agents, or fordiagnostic imaging.

In one embodiment of the instant invention, a method of measuring bloodflow in a subject comprises the following steps:

-   -   a) localizing a detectable amount of the FA composition of the        invention, to a tissue of interest;    -   b) detecting a signal from said radiolabeled FA composition in a        tissue of interest within about 1 minute and about 5 minutes        after administration;    -   c) imaging a tissue of interest and;    -   d) determining the rate of blood flow in a tissue of interest.

The present invention further provides a method of measuring metabolismin a subject comprising the following steps:

-   -   a) localizing a detectable amount of the FA composition of the        invention, to a tissue of interest and;    -   b) detecting a signal from said radiolabeled FA composition in a        tissue of interest within about 30 minutes and about 120 minutes        after administration;    -   c) imaging a tissue of interest and;    -   d) determining the rate of metabolism in a tissue of interest.

Another embodiment of the present invention provides a method forretaining a fatty acid composition of the invention in a tissue ofinterest, comprising the steps of:

-   -   a) localizing a detectable amount of the composition to the        tissue;    -   b) retaining the composition, or a metabolic derivative thereof,        in the tissue by reducing transport and back-diffusion of the        composition; and    -   c) detecting the composition or the metabolic derivative in the        tissue.

In another embodiment of the present invention, a method for retaining afatty acid composition of the invention in a tissue of interest,comprising the steps of:

-   -   a) localizing a detectable amount of the composition to the        tissue;    -   b) retaining the composition, or a metabolic derivative thereof,        in the tissue by reducing dehydrogenation of the composition;        and    -   c) detecting the composition or the metabolic derivative in the        tissue.

The present invention further provides a method for retaining a fattyacid composition of the invention in a tissue of interest, comprisingthe steps of:

-   -   a) localizing a detectable amount of the composition to the        tissue;    -   b) retaining the composition, or a metabolic derivative thereof,        in the tissue by reducing hydroxylation of the composition; and    -   c) detecting the composition or the metabolic derivative in the        tissue.

Another embodiment of the present invention provides a method forretaining a fatty acid composition of the invention in a tissue ofinterest, comprising the steps of:

-   -   a) localizing a detectable amount of the composition to the        tissue;    -   b) retaining the composition, or a metabolic derivative thereof,        in the tissue by reducing ketoacyl formation of the composition;        and    -   c) detecting the composition or the metabolic derivative in the        tissue.

Yet another embodiment of the present invention provides a method forretaining a fatty acid composition of the invention in a tissue ofinterest, comprising the steps of:

-   -   a) localizing a detectable amount of the composition to the        tissue;    -   b) retaining the composition, or a metabolic derivative thereof,        in the tissue by reducing ketoacetyl elimination of the        composition; and    -   c) detecting the composition or the metabolic derivative in the        tissue.

Numerous methods by which one may use the present invention in imaging,measurements of blood flow, and FA metabolism are contemplated, usedsingularly, or in combination with other imaging modalities. Forexample, cardiac imaging modalities generally measure two parameters:blood flow (perfusion) and myocardial viability (metabolism). Thesemeasurements are useful in numerous cardiac diseases. Animal models ofmeasuring blood flow often use microspheres (Miller, D. D., et al.(1988) Circ. Res. 63: 681-692), however their use is precluded inhumans, as microspheres can block circulation. Traditional methods ofobserving myocardial perfusion and metabolism have utilized twodifferent tracers each separately used to measure either blood flow ormetabolism. Widely used markers for blood flow are exemplified by²⁰¹Thallium (²⁰¹Tl), ^(99m)Technetium (^(99m)Tc), ¹³N-Ammonia, and⁸²Rubidium. However, unlike ²⁰¹Tl, ^(99m)Tc, ¹³N-Ammonia, and⁸²Rubidium, the uses of which are only limited to measurements ofperfusion, certain FA tracers are able to measure both flow andmetabolism (Poe, N. D. et al. (1977) Radiology124: 419-424; van derWall, E. E. et al. (1980) Eur. J. Nucl. Med. 5: 401-405; Kairento, A. L.et al. (1988) Nucl. Med. Biol. 15: 333-338; Kawamoto, M. et al. (1994)J. Nucl. Cardiol. 1: 522-8; Kobayashi, H. et al. (1997) J. Nucl. Med.39: 1117-1122; Miller, D. D., et al. (1988) Circ. Res. 63: 681-692).

Similarly, the instant invention can be used in a variety of methods toanswer a specific diagnostic question. By way of example, methods caninclude administration of the invention under conditions of rest and/orstress induced by exercise, disease states, or pharmacological agents.Likewise, the radiolabeled FAs having a cyclic organic substituent canalso be used in sequential imaging experiments, depending on theradioisotope used. Blood flow to an organ of an animal can be determinedwithin 1 second to about 10 minutes, preferably between about 1 minuteand about 5 minutes after the radiolabeled cyclic substituted FA isadministered to the animal. Metabolism by the organ of interest can bedetermined within a time period between about 10 minutes to about 24hours, preferably between about 30 minutes and about 120 minutes afteradministration of the radiolabeled FA into the bloodstream of theanimal.

Nuclear cardiac imaging using the present invention can be used todetect a wide variety of cardiac derangements. One of sufficient skillin the art will recognize that cyclic substituted, radiolabeled FAs canbe used singularly as a marker for blood flow and for cardiacmetabolism, or may be used in combination with another tracer. Suchtracers include, but are not limited to ¹³N-Ammonia,⁵⁷Co-Cyanocobalamin, ⁵⁹Fe-Ferrous Citrate, ¹⁸F-Fluorodeoxyglucose,⁶⁷Ga-Gallium Citrate, ¹¹¹In-Indium Oxyquinoline, ¹¹¹n-Indium Pentetate,¹¹¹In-Indium Pentetreotide, ¹¹¹In-Indium Satumomab Pendetide,Radioiodinated Iobenguane, ¹²³I-Iodohippurate Sodium, ¹³¹I-IodohippurateSodium, ¹²³I-lofetamine, ¹²⁵I-Iothalamate Sodium, ⁸¹Krypton,¹¹C-Methionine, RadioiodinatedAlbumin, ⁸²Rubidium, Sodium ⁵¹Chromate,Sodium ¹⁸Fluoride, Sodium ¹²³Iodide, Sodium ¹³¹Iodide, Sodium^(99m)-Pertechnetate, ^(99m)Tc-Albumin, ^(99m)Tc-Albumin (Aggregated),^(99m)Tc-Albumin (Colloidal), ^(99m)Tc Arcitumomab, ^(99m)Tc-Bicisate,⁹⁹m Tc-Disofenin, ^(99m)Tc-Exametazime, ^(99m)Tc-Gluceptate,^(99m)Tc-Lidofenin, ^(99m)Tc-Mebrofenin, ^(99m)Tc-Medronate,^(99m)Tc-Mertiatide, ^(99m)Tc-Nofetumomab Merpentan,^(99m)Tc-Oxidronate, ^(99m)Tc-Pentetate, ^(99m)Tc-Pyrophosphate,^(99m)Tc-(Pyro- and trimeta-)Phosphates, ^(99m)Tc Sestamibi, ^(99m)TcSuccimer, ^(99m)Tc-Sulfur (Colloidal), ^(99m)Tc-Teboroxime,^(99m)Tc-Tetrofosmin, ²⁰¹Thallous Chloride, ¹²⁷Xenon, ¹³³Xenon. It willbe apparent that not all of the aforementioned tracers are suitable forcardiac imaging and that use of the instant invention is not necessarilylimited to those related to cardiovascular disease.

The tissues of interest can be any tissues that utilize FAs as a sourceof energy. The tissues can be, but are not limited to, cardiac tissue,brain, liver, bone, spleen, lung, blood, kidney, gastrointestinal,muscle, and adrenal tissue. A preferred embodiment of the presentinvention is cardiac tissue. Another preferred embodiment can alsoencompass liver tissue.

Cardiac diseases or disease states that can be monitored usingradiolabeled FAs having cyclic organic substituents include, but are notlimited to, acute myocardial infarction, unstable angina, chronicischemic heart disease, coronary artery disease, myocarditis, dilated,hypertrophic, and restrictive cardiomyopathies, congenital heartdiseases, hypertensive heart disease, post-transplant heart disease,allograft vasculopathies, valvular heart disease, and pharmacologicallyinduced conditions such as doxorubicin cardiotoxicity. It iscontemplated that methods of use of radiolabeled FAs exemplified by thepresent invention will be modified according to the particular diseaseexamined.

The instant invention can also be used in numerous non-cardiac diseasestates described herein, such as: abscess and infection; biliary tractblockage; blood volume studies; blood vessel diseases; blood vesseldiseases of the brain; bone diseases; bone marrow diseases; braindiseases and tumors; cancer and neoplasms; colorectal disease; diabetes;disorders of iron metabolism and absorption; heart disease; heart muscledamage such as infarction and ischemia; impaired flow of cerebrospinalfluid in brain; kidney diseases; liver diseases; lung diseases;parathyroid diseases and/or parathyroid cancer; pernicious anemia and/orimproper absorption of vitamin B₁₂ from intestines; red blood celldiseases; salivary gland diseases; spleen diseases; stomach disordersand intestinal bleeding; tear duct blockage, thyroid diseases and/orthyroid cancer, urinary bladder diseases. A preferred embodiment of thepresent invention is its use in detecting cardiac myopathies bymeasuring blood flow and FA metabolism. It is understood that diagnosisof the aforementioned diseases will often require the use of otherradiotracers, also described above. It is apparent that use of theinstant invention is not only limited to detection of diseased states,but also for diagnostic imaging in healthy subjects.

One of ordinary skill in the art will appreciate the numerous methods ofdetecting cardiac imaging agents exemplified by the present invention,as well as understand that different radionuclides will requiredifferent detection techniques. Positron Emission Tomography (PET) andSingle Photon Emission Computed Tomography (SPECT) are imagingtechniques in which a radionuclide is synthetically introduced into amolecule of potential biological significance, such as a radiolabeled FAindicated by the instant invention. The subsequent uptake of theradiotracer is measured over time and used to obtain information aboutthe physiological process of interest. While PET and SPECT rely onsimilar principles to produce their images, important differences ininstrumentation, radiochemistry, and experimental applications aredictated by inherent differences in their respective physics of photonemission.

Unstable nuclides that possess an excess number of protons may take oneof two approaches in an effort to reduce their net nuclear positivity.In one radioactive decay scheme, a proton is converted to a neutron anda particle called a positron is emitted (Hoffman, E. J., and Phelps, M.E. New York: Raven Press; 1986: 237-286; Sorenson, J. A., and Phelps, M.E. Philadelphia: W. B. Saunders; 1987). Of identical mass but oppositecharge, positrons are the antimatter equivalent of electrons. Whenejected from the nucleus, a positron collides with an electron,resulting in the annihilation of both particles and the release ofenergy. Two γ photons are produced, each of equivalent energy andopposite trajectory (generally 180° apart).

The unique spatial signature of back-to-back photon paths is exploitedby PET scanners in locating the source of an annihilation event, amethod known as coincidence detection (Hoffman, E. J., and Phelps, M. E.New York: Raven Press; 1986: 237-286; Links, J. M. New York: RavenPress; 1990: 37-50). PET (and SPECT) scanners employ scintillationdetectors made of dense crystalline materials (e.g., bismuth germaniumoxide, sodium iodide, or cesium fluoride), that capture the high-energyphotons and convert them to visible light. This brief flash of light isconverted into an electrical pulse by an adjacent photomultiplier tube(PMT). The crystal and PMT together make up a radiation detector. A PETcamera is constructed such that opposing detectors are electronicallyconnected. Thus, when separate scintillation events in paired detectorscoincide, an annihilation event is presumed to have occurred at somepoint along an imaginary line between the two. This information is usedto reconstruct images using the principles of computed tomography.Conversely, single events are ignored. Although it is conceivable thattwo unrelated photons from spatially separate annihilation events mightreach opposing detectors in unison, these accidental coincidences aremuch less frequent than true ones. Nevertheless, random coincidencesconstitute a source of background noise in PET images (Hoffman E J etal. J Comput Assist Tomogr (1981); 5:391-400; Hoffman, E. J., andPhelps, M. E. New York: Raven Press; (1986): 237-286; Links, J. M. NewYork: Raven Press; (1990): 37-50).

The skilled artisan will recognize the intrinsic limitations of PETderive from the nature of positron decay and the principle ofcoincidence detection. Specifically, PET recognizes the site of positronannihilation, which does not necessarily coincide with the site ofradioactive decay. Annihilation often occurs some distance away from thepositron's origin. The distance separating these two events, decay andannihilation, depends on the average kinetic energy of the positron asit leaves the nucleus, and varies according to the specific isotopeinvolved (Phelps M E, et al. J Nucl Med (1975); 16: 649-652). Inaddition, if the positron is not entirely at rest at annihilation,photons will be emitted at an angle slightly different than 180°. Takentogether, remote positron annihilation and photon non-colinearity placea theoretical limit on PET's achievable spatial resolution (Links J M.New York: Raven Press; (1990): 37-50).

In another embodiment of the present invention, isotopes that decay byelectron capture and/or γ emissions can be directly detected by SPECT.Certain proton-rich radionuclides, such as ¹²³I and ^(99m)Tc, mayinstead capture an orbiting electron, once again transforming a protonto a neutron (Sorenson J A, and Phelps M E. Philadelphia: W. B.Saunders; 1987). The resulting daughter nucleus often remains residuallyexcited. This meta-stable arrangement subsequently dissipates, therebyachieving a ground state and producing a single γ photon in the process.Because γ photons are emitted directly from the site of decay, nocomparable theoretical limit on spatial resolution exists for SPECT.However, instead of coincidence detection, SPECT utilizes a techniqueknown as collimation (Jaszczak R J. Boca Raton: CRC Press; (1991):93-118). A collimator may be thought of as a lead block containing manytiny holes that is interposed between the subject and the radiationdetector. Given knowledge of the orientation of a collimator's holes,the original path of a detected photon is linearly extrapolated and theimage is reconstructed by computer-assisted tomography.

It is contemplated that use of the instant invention will requireprocessing and analysis of data obtained from imaging modalities such asbut not limited to PET or SPECT. The following mathematical algorithmscan determine the rate of uptake of novel radiolabeled FA analogs havinga cyclic organic substituent. Other mathematical models can also besimilarly employed and/or modified to determine the rate of blood flowand metabolic activity (Sokoloff, L. et al (1977) J. Neurochem. 28:879-916; Elmaleh, D. R. et al. (1994) J. Nucl. Med. 35: 496-503;Marshall, R. C. et al. (1998) Am. J. Physiol.275: H668-H679; Wieler, H.et al. (1990) Nuc. Med. Commun. 11: 865-878). The collected early andlate images in tissues of interest can be manipulated to obtain valuesthat may indicate abnormalities.

Mathematical modeling can be used to assess the rate of uptake of FAanalogs of the present invention. There is a reproducible relationshipbetween the net extraction fractions of native FA and FA analog.Assuming the uptake and metabolism of native FA is in a steady state,the rate of metabolism, defined as R for purposes of this application,can be deduced from the Fick equation:R=E _(n) *F*Ca (mol/min/g)where E_(n)=net extraction fraction

-   -   -   F=blood flow (ml/min/g), which can be estimated from the            early images.

    -   Ca=arterial concentration of FA (mol/ml).

The assumption is such that at steady state, the rate at which FA leavesthe tissue through oxidation must be equal to the rate at which FA isentering the tissue from the blood. The net extraction of a substancemetabolized by the heart in a steady state can be determined from thearterial or arterialized blood and venous blood concentrations of FAs.

A relationship between the net extraction of FA analog and FA, E_(n)(FAanalog), can be used to calculate the rate of FA metabolism, R. We canwrite this relationship asL=E _(E) /E _(n)(FA analog)

-   -   L here plays the same role as the “lumped constant” in        Sokoloff's model for deoxyglucose metabolism (Sokoloff, L. et        al (1977) J. Neurochem. 28: 879-916). The difference between the        two models is that L may not be constant. ‘L’ can vary with        blood flow, free fatty acid concentration, FFA composition, or        other physiological parameters. The behavior of L is best        understood by performing a series of experiments to determine        the relationship between E_(n) and E_(n)[FA analog] in different        physiological states.

Basic modeling assumptions are such that 1) fatty acid uptake andmetabolism is in a steady state. In other words, all the FA pools andreaction products have constant concentrations. This assumes that thenet rate at which FA is removed from the blood equals the rate of FAcatabolism. (2) Labeled FA analog is generally present in trace amounts,so that its presence does not alter the FA steady state. The varioustransport and chemical reaction rates for FA analog can then be expectedto depend linearly on the concentrations of the FA analogs. Thismodeling approach accounts for capillary-tissue exchange as well as forintracellular metabolism of the administered FA analog. Intracellularmetabolism of native fatty acids is assumed to create gradients, whichregulate the access of FA analog to the intracellular space. Coupled tothese metabolically induced gradients are the effects of blood flow andcapillary transport properties. In this approach, flow and transportmechanisms are included in capillary tissue exchange while the metabolicprocesses are included in intracellular metabolic parameters.

In general, the transport of any substance between capillary and tissuedepends on two factors: (i) Intrinsic capillary transport rate for afreely diffusing substance (or the permeability surface area product;for carrier mediated transport, it is a function of “apparent”Michaelis-Menten parameters that describe the transport process). In a“microscopic” modeling approach, a protein-substrate complex wouldcharacterize each barrier, but here they are lumped together into asingle “apparent” rate parameter (k). (ii) Local blood flow per ml oftissue. Blood flow and transport rate can be thought to describe twocompeting processes, one carrying molecules through the capillary wall,the other carrying them out the venous end of the capillary. These tworates are combined to give extraction fraction E, the fraction ofmolecules entering a capillary bed, which pass through the wall of thecapillary at least once. As used here, E is the one-way (unidirectional)extraction fraction, not the net extraction fraction.

It is assumed that an FA analog cannot undergo β-oxidation, and hence,is either trapped in mitochondria or incorporated in the triglyceridepool. The triglyceride pool turns over very slowly. With respect to FAanalog, the concentration of FA analog in myocardial tissue, followingIV injection, can reach a plateau after ten to fifteen minutes. Thesimplest model consistent with the measured tissue curves and thebiochemical data is one in which tissue is described by twocompartments, a precursor FFA pool and a metabolically trapped pool,both driven in response to the FA analog concentration in the bloodplasma.

Because FA analogs cannot undergo β-oxidation and because the turnoverrate for the triclyceride pool is slow, the plateau of the tissue curvecan reflect both components. Measurements with FA analogs can onlydetermine the rate at which FA analog passes through the committed stepto acyl-CoA. Accordingly, the steady state rate for incorporation intotriglyceride plus β-oxidation can be measured. This situation isanalogous to the result that would be obtained if Fick-typeatrial/ventricular measurements with a native fatty acid such aspalmitate could be made.

Based on the considerations outlined above, an operational equationcapable of describing the PET measurements in terms of the blood flow isdescribed, F being the unidirectional extraction fraction, E as the netextraction fraction, E_(n), and the rate K, with which FA analog iscleared from the “precursor pool”. The rate constant K is the sum of tworates, K₂ the rate of back-diffusion, and K₃, the rate of activation ofFA analog to the CoA form. Neglecting tracer in blood, the operationalequation is given asC(t)=F*[(E=E _(n))*Exp(−K*t)+E _(n)] (*) Ca (t)Where C(t) is the tissue concentration at time t,

-   -   Ca(t) is the plasma concentration at time t and    -   (*) represents the mathematical operation of convolution.

For the model of this invention, E_(n)=E*K3/K2+K3. This expression has asimple interpretation—i.e., E is the probability a FA analog moleculewill leave the blood on a single capillary transit, K3/(K2+K3) is theprobability that a FA analog molecule entering the tissue will bemetabolically trapped. No assumptions were made in the derivation of theoperational equation limiting the value of E, other than it be in therange (0.1). It should also be noted that the operational equation isformally similar to that of Sokoloff.

Under the assumption that a steady state was established with respect tothe consumption of FA analog, where the plasma FA content was theconcentration of FA analog, then the analog's metabolic rate would beF*E_(n)[FA analog]. Therefore, the quantity F*E_(n) can be interpretedas the analog utilization rate to a unit quantity of FA analog in steadystate.

Analysis of the operational equation of the method shows that as t (or,the plateau concentration is given by F*E_(n)*∫ of the plasmaconcentration curve). Thus, the tissue concentration at the plateau isdirectly proportional to the metabolism of the analog.

A better understanding of the instant invention and of its manyadvantages will be facilitated via the following examples, which furtherdescribe the invention and given by way of illustration. The examplesthat follow are not to be construed as limiting the scope of theinvention in any manner. In light of the present diclosure, numerousembodiments within the scope of the claims will be apparent to those ofordinary skill in the art.

EXAMPLES Example 1 Synthesis of ¹⁸F-labeled3,4-cyclopropyl-heptadecanoic acid ([¹⁸F]-FCPHA)

Synthesis of [¹⁸F]-FCPHA was performed using the following method. PCC(pyridinium chlorochromate; 24.8 g, 115.2 mmol) was slowly added inportions to a solution of 6-benzyloxy-1-hexanol (20 g, 96 mmol) in 200ml of methylene chloride at room temperature (25° C., RT). The blackmixture was stirred for 2 hours, and filtered through 50 g of silicagel. The solvent was removed and the crude oil was separatedchromatographically on silica gel using a mixture of hexane:ethylacetate at a ratio of 90:10. This yielded the aldehyde equivalent,6-benzyloxy-1-hexanal, at a scale of 14 g (70%).

Under a nitrogen atmosphere, octyl bromide (12 g, 115 mmol) in anhydrousether (60 mL) was added to magnesium metal (2.8 g, 116 mmol) in 10 ml ofether at a rate as to maintain gentle reflux. After addition, thereaction mixture was stirred for 1 hour and then 6-benzyloxyhexanal (12g, 58 mmol) in 20 ml of ether was added dropwise to the reaction mixtureat RT. The mixture was stirred for 4 hours and poured over ice water.Acidification was achieved with 10% hydrochloric acid (HCl), thenextracted with ether. The combined extracts were washed with brine,dried, and the solvent removed. Chromatography of the crude oil onsilica gel using hexane:ethyl acetate at a ratio of 80:20 yielded 16.5g, or 89%, of the alcohol. The chemical shift of the compound, detectedby ¹H-NMR, was as follows: ¹H NMR (CDCl₃) δ 0.89 (t, 3H, CH₃), 1.2-1.7(m, 22H, CH₂), 3.45 (m, 2H, CH₂O), 3.55 (m, 1H, CH—O), 4.55 (s, 2H,CH₂—O), 7.35 (m, 5H, benzyl).

The resulting compound, 1-benzyloxy-6-tetradecanol (16 g), washydrogenated overnight using 100 mg of 10% palladium on activated carbon(Degussa AG; Frankfurt, Germany) in 100 ml of methanol to yield1,6-tetradecandiol (11.1 g, 4.85 mmol). This diol compound, in 100 ml ofmethylene:pyridine (9:1), was treated with trityl chloride (13.5 g, 4.85mmol). The mixture was stirred for 4 hours, then washed with ice-cold10% HCl until the aqueous phase was acidic. Washing in brine thenfollowed this until the aqueous phase was neutral. Solvent removal andchromatography on silica gel in hexane:ethylacetate (80:20) of the oilgave 20.1 g (87%) of the trityl alcohol. The chemical shift of thecompound, detected by ¹H-NMR, was as follows: ¹H-NMR (CDCl₃) δ 0.89 (t,3H, CH₃), 1.2-1.7 (m, 22H, CH₂), 3.05 (m, 2H, CH₂O), 3.55 (m, 1H, CH—O),3.55 (m, 1H, CH—O), 7.2-7.5 (m, 15H, benzyl).

The resulting compound, 1-trityl-6-tetradecanol (20 g, 42.3 mmol), wasslowly mixed with 60% sodium hydride (2 g, 50 mmol) and benzyl bromide(7.2 g, 42.3 mmol) in dry dimethylformamide (DMF; 100 ml) in a waterbath set at RT. The mixture was stirred for 4 hours at RT and pouredinto ice water, then extracted with ether. Chromatography of the crudeoil on silica gel using hexane:ethyl acetate (90:10) yielded 19 g (34mmol), or 80% of the diether compound, 6-benzyloxy-1-trityl-tetradecanediether. This diether compound was mixed with 100 mg of p-toluenesulfonic acid in methanol (100 L) at RT for 4 hours. Sodium bicarbonate(100 mg) was added and the solvent removed by rotary evaporation. Theoil was separated by chromatography on silica gel in hexane:ethylacetate (80:20) yielded 7.5 g, or 90% of the alcohol. The chemical shiftof the resulting compound, 6-benzyloxy-1-tetradecanol, is as follows:¹H-NMR (CDCl₃) δ 0.89 (t, 3H, CH₃), 1.2-1.7 (m, 22H, CH₂), 3.38 (m, 2H,CH₂O), 3.55 (m, 1H, CH—O), 4.7 (s, 2H, CH₂—O), 7.35 (m, 5H, benzyl).

PCC (9.7 g, 45 mmol) was added in portions to a solution containing6-benzyloxy-1-tetradecanol (7.5 g, 30 mmol) in methylene chloride (100ml) at RT. The black mixture was stirred for 2 hours, then filteredthrough a bed of silica gel using hexane:ethyl acetate (90:10). Thecorresponding aldehyde, 6-benzyloxy-tetradecanal, was produced at ayield of 6.1 g (81%). This aldehyde compound was mixed withcarbethoxymethylene triphenylphosphorane (10 g, 29 mmol) in 100 ml ofmethylene chloride at RT. The mixture was allowed to stir overnight,after which the solvent was removed and replaced with hexane. Solidprecipitates were removed by filtration. Following chromatography onsilica gel using hexane:ethyl acetate at a ratio of 90:10, thecorresponding ester was detected at 8.4 g scale (90%). The chemicalshift of the ester, 8-benzyloxyhexadec-2-enoate ethyl ester, is asfollows: ¹H-NMR (CDCl₃) δ 0.89 (m, 6H, CH₃), 1.2-1.5 (m, 20H, CH₂), 2.2(m, 2H, CH₂—C═), 3.35 (m, 2H, CH₂O), 4.2 (q, 2H, MeCH₂—O), 4.55 (dd, 2H,CH₂—O), 5.8 (d, 1H, CH═C), 6.95 (d,t 1H, CH═C), 7.35 (m, 5H, benzyl).

DIBAL (diisobutylaluminum hydride; 8 ml, 44 mmol) in 40 ml hexane, wasadded dropwise to the ester compound 8-benzyloxyhexadec-2-enoate ethylester (8.4 g, 22 mmol) in 40 ml methylene chloride at −78° C. Themixture was allowed to reach 0° C. and 5 ml of ethyl acetate was addeddropwise, followed by ice. The resultant slurry was acidified with 10%HCl, followed by ether extraction. The combined ether extracts werewashed with brine, dried, and the solvent was removed. Separation of thecrude oil by silica gel chromatography using hexane:ethyl acetate(80:20) yielded 6.7 g, or 90% of the unsaturated primary alcohol.

Addition of the cyclopropyl moiety was performed essentially asdescribed by Charette and coworkers (Charette et al, J. Org. Chem. 199560: 1081). A 100-ml round bottom flask containing 40 ml of dry methylenechloride was cooled to −25° C. and placed under a nitrogen atmosphere.To this flask, 3.8 ml of diethyl zinc, followed by 3.9 ml of1,2-dimethoxyethane (DME), was added. Diiodomethane (6 ml) was addeddropwise to the solution, while maintaining the reaction temperaturebetween −25° C. and −10° C. This solution was then added by double-endedneedle, to a solution containing 8-benzyloxyhexadec-2-enol (2 g, 5.8mmol), dioxaborolane (made from (+)N,N,N′,N′-tetramethyl-L-tartaramideand butylboronic acid), and 300 mg of 4 Å molecular sieves in methylenechloride (40 ml) under nitrogen at −40° C. and −30° C. The reactionmixture was stirred for 2 hours at −25° C., then allowed to warm to 0°C. Saturated ammonium chloride in an 80 ml volume was added to themixture, then stirred at RT for 16 hours. Solid matter was removed byfiltration and the layers were separated. The aqueous layer wasextracted with methylene chloride, while organic layers were pooled,washed with water, and dried. The crude oil was separated by silica gelchromatography in hexane:ethyl acetate (90:10), yielding 1.9 g (90%) ofthe cyclopropyl alcohol. This reaction was repeated twice. The chemicalshift of the molecule, 8-benzyloxy-2,3-cyclopropylhexadecanol, is asfollows: ¹H-NMR (CDCl₃) δ 0.3 (m, 2H, ring CH₂), 0.5 (m, 1H, CH), 0.75(m, 1H, CH), 0.89 (t, 3H, CH₃), 1.2-1.6 (m, 22H, CH₂), 3.35 (m, 1H,CHO), 3.45 (m, 2H, CH₂—O), 4.55 (s, 2H, CH₂—O), 7.35 (m, 5H, benzyl).Addition of dioxaborolane in this reaction will form the S,S isomer asthe predominant species. The reaction can be performed withoutdioxaborolane to yield the racemic (R,R; S,S) mixture.

Iodine (6 g, 24 mmol) was added in portions to a solution containing8-benzyloxy-2,3-cyclopropylhexadecanol (5.7 g, 15.8 mmol) andtriphenylphosphine (6.2 g, 23.7 mmol) in 100 ml of dimethylformamide.The dark red mixture was combined with 100 ml of 10% sodium thiosulfateand extracted with ether. The combined extract was washed with water,brine, and the solvent was subsequently removed. Silica gelchromatography in hexane:ethyl acetate (95:5) yielded 4.5 g (60%) of thealkyl iodide. This alkyl iodide,8-benzyloxy-1-iodo-2,3-cyclopropylhexadecane (4.5 g, 9.6 mmol), andsodium cyanide (0.95 g, 19.2 mmol) in 60 ml of dimethyl sulfoxide(DMSO), was heated to 80° C. for 2 hours. The mixture was poured into100 ml of water, followed by ether extraction. The combined extract waswashed with water, dried, and solvent removed. The crude oil wasseparated by silica gel chromatography in hexane:ethyl acetate (80:20)and yielded 3.0 g (87%) of the corresponding nitrile compound. Chemicalanalysis by ¹H-NMR resulted in: ¹H NMR (CDCl₃) δ 0.3 (m, 2H, ring CH₂),2.4 (m, 2H, CH₂—CN), 3.35 (m, 1H, CHO), 4.55 (s, 2H, CH₂—O), 7.35 (m,5H, benzyl).

A mixture containing the above-described nitrile compound,9-benzyloxy-2,3-cyclopropylhexadecane nitrile (3.0 g, 8.1 mmol), 600 mgof potassium hydroxide (KOH), and 3 drops of water in 40 ml ethyleneglycol, was heated for 6 hours at 170° C. Once cooled, the mixture wasdiluted with 80 ml of 10% HCl, then extracted with ether. The combinedextract was dried and the solvent was removed. The crude carboxylic acidwas treated with diazomethane (generated fromN-methyl-N′-nitro-N-nitrosoguanidine and 40% KOH in ether). The reactionmixture was stirred for 1 hour before solvent removal. Chromatography ofthe crude oil on silica gel with hexane:ethyl acetate (80:20) gave 1.9g, or 60%, of the corresponding ester,9-benzyloxy-3,4-cyclopropylheptadecanoate methyl ester (4.7 mmol). Thisester was hydrogenated overnight with 25 mg of 10% palladium onactivated charcoal in 50 ml of methanol. Hydrogenation yielded 1.3 g, or89% of the hydroxy ester. Chemical analysis resulted in the following:¹H-NMR (CDCl₃) δ 0.30 (d,d, 2H, ring CH₂), 0.53 (m, 1H, CH), 0.75 (m,1H, CH), 0.89 (t, 3H, CH₃), 1.2-1.6 (m, 22H, CH₂), 2.23 (m, 2H, CH₂—CO),3.58 (m, 1H), CHO), 3.69 (s, 3H, CH₃).

The hydroxy ester, 9-hydroxy-3,4-cyclopropylheptadecanoate methyl ester(0.5 g, 1.6 mmol), and methane sulfonyl chloride (0.2 g, 1.76 mmol), and10 mg of DMAP (4-dimethylaminopyridine) in 20 ml of methylenechloride:pyridine (90:10), was stirred for 2 hours. The mixture waswashed with 10% HCl until the aqueous layer was acidified, then washedagain with 10% NaHCO₃. Chromatographic analysis of the crude oil onsilica gel, in methylene chloride:methanol (95:5) gave 0.5 g (83%) ofthe mesylate ester, 9-hydroxy-3,4-cyclopropylheptadecanoate methylester.

The radiolabel ¹⁸F, contained in water (60 mCi, 1 ml), was added to avial containing 10 mg of Kryptofix-222(4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane; Merck,Whitehouse Station, N.J.) and 4 mg of K₂CO₃. Water was removed using astream of nitrogen at 115° C., folowed by the three rounds of additionof acetonitrile (2 ml). The mesylate ester,9-hydroxy-3,4-cyclopropylheptadecanoate methyl ester (10 mg) in 1 ml ofacetonitrile was combined with the radiolabel mixture and heated for 10minutes in 120° C. This yielded the ¹⁸F-labeled ester at 80% yield aftersilica Sep-Pak purification (Waters Corporation, Milford, Mass.) inhexane:ethyl acetate (85:15). The labeled ester was placed in a vial andthe solvent was removed. It was replaced with 0.1 ml of 1M lithiumhydroxide (LiOH) and 0.3 ml of methanol. The reaction vial was heated to60° C. for 20 minutes. Methanol was removed by nitrogen stream at 60° C.The solution was acidified in 10 ml of 10% HCl, and the mixture wasextracted twice with 10 ml of ether. The combined ether extract waspassed through a silica Sep-Pak chromatography cartridge and solvent wasremoved, affording 13 mCi of the final product,9-[¹⁸F]-fluoro-3,4-cyclopropylheptadecanoic acid. Radio thin-layerchromatography (TLC) showed 94% radiochemical purity (silica gel plate,25:75 ethyl acetate/hexane, R_(f)=0.40) with the remaining activity asunhydrolyzed ester (silica gel plate, 15:85 ethyl acetate/hexane,R_(f)=0.80). A schematic representation of the organic synthesisdescribed above is shown in FIG. 1. The labeled fatty acids wereformulated in 10% ethanol in saline for rat studies and 4% BSA in salinefor monkey studies (sterile filtered through a 0.22 μm Milliporefilter).

Example 2 Biodistribution of ¹⁸F-Labeled 3,4-cyclopropyl-heptadecanoicacid([¹⁸F]-FCPHA)

The initial myocardial behavior of [¹⁸F]-FCPHA was essentially similarto that of normal FAs, as evidenced that this analog concentrates in thesame metabolic pools and has the same subcellular distribution in therat heart, where it is found in the mitochondria. Male Sprague-Dawleyrats weighing approximately 300-350 grams, were injected withoutanesthesia with 40 μCi of [¹⁸F]-FCPHA in a total volume of 200 μl. Theradiolabeled FA was injected into the tail vein and the ratssubsequently sacrificed at 5 minutes and 60 minutes post-injection. Theorgans were excised and counted in a gamma counter. Accumulation of theFA in various organs in % DPG (dose per gram) is detailed in Table 1.

TABLE 1 Accumulation of ¹⁸F-FCPHA in Rat Tissue Tissue % DPG (5 min) %DPG (60 min) Heart 1.55 ± 0.66 1.43 ± 0.14 Lung 0.47 ± 0.12 0.33 ± 0.16Liver 1.34 ± 0.26 0.87 ± 0.15 Bone 0.16 ± 0.03 0.70 ± 0.39 Blood 0.064 ±0.008 0.080 ± 0.019 Spleen 0.55 ± 0.30  0.34 ± 0.085 Kidney  0.41 ±0.097 0.34 ± 0.08 Adrenal  0.22 ± 0.061 0.30 ± 0.16 Stomach  0.12 ±0.067  0.15 ± 0.038 Gastrointestinal Tract  0.19 ± 0.040  0.24 ± 0.039Testes 0.020 ± 0.003 0.011 ± 0.004 Brain 0.06 ± 0.01 0.05 ± 0.02 Muscle 0.25 ± 0.009 0.097 ± 0.075Bone accumulation of the fluorinated FA measured de-fluorination of thecompound and its subsequent accumulation. To confirm imaging resolution,the following ratios were determined at 5 and 60 minutes post-injection(Table 2).

TABLE 2 Imaging Resolution of ¹⁸F-FCPHA Tissue Ratio Ratio at 5 minutesRatio at 60 minutes Heart-to-blood 25.8 20.4 Heart-to-lung 3.3 4.6Table 3 shows the biodistribution of the β-methyl analog, [¹⁸F]FBMHA, at5 and 60 minutes after intravenous administration in rats (5 per timepoint). At 5 minutes after injection, accumulation of radioactivity inthe heart was 2.56% dose per gram, with nearly an equal amount ofradioactivity in the kidneys. Tracer activity in the blood at 5 minuteswas 1.02% DPG, and remained relatively high (0.58% DPG) after 60minutes. Most of the radioactivity accumulated in the liver. Theheart-to-blood ratio of 2.6 at 5 minutes did not significantly changeafter 60 minutes. There was bone accumulation of 0.52% DPG at 5 minutes,which increased to 2.36% DPG at 60 minutes, indicating extensivedefluorination. Biodistribution of [¹⁸F]FBMHA in rats was repeated usinga 4% Tween-80/saline formulation and similar results were obtained.

TABLE 3 Tissue distribution of [¹⁸F]FBMHA in Sprague-Dawley rats Organ 5minutes 60 minutes Blood 1.02 ± 0.22 0.58 ± 0.27 Heart 2.56 ± 0.76 1.69± 0.13 Lung 1.25 ± 0.18 0.58 ± 0.17 Liver 8.88 ± 2.97 4.17 ± 1.49 Kidney2.62 ± 0.86 1.66 ± 0.61 Muscle 0.66 ± 0.22 0.41 ± 0.10 Bone 0.52 ± 0.162.36 ± 0.78 Brain 0.25 ± 0.07 0.27 ± 0.03Table 4 represents heart-to-blood ratios in rats 60 minutes afteradministration of labeled [¹⁸F]FCPHA compared to other β-methyl analogs[¹⁸F]FBMHA, [¹¹C]BMHA, and [¹²⁵I]BMIPP.

TABLE 4 Heart to blood ratios of various β-methyl analogs [¹⁸F]FCPHA[¹⁸F]FBMHA [¹¹C]BMHA [¹²⁵I]BMIPP Ratio 20:1 3:1 10:1 3:1

Example 3 Comparison of the Biodistribution of R,R and S,S Diastereomerswith Racemic Mixtures of ¹¹C or ¹⁴C-Labeled 3-methyl-heptadecanoic Acid

The following data compare the biodistribution of1-[¹¹C]-3(S)-methylheptadecanoic acid (the ‘S’ isomer) andI-[¹¹C]-3(R)-methylheptadecanoic acid (the ‘R’ isomer). Also compared isthe racemic mixture, 1-[¹⁴C]-3(R,S)-methylheptadecanoic acid, labeledwith ¹⁴C. Measurement of the % DPE (dose per gram) at 5 minutes and 30minutes revealed that the ‘S’ isomer has higher heart uptake and higherheart-to-blood ratios, indicating that the ‘S’ isomer is preferable thanthe ‘R’ isomer or the racemic mixture as a potential heart imagingagent. Biodistribution was performed in six rats for each compound andtime point. The 5-minute distribution data are contained in Table 5,while the 30-minute data are contained in Table 6. These unexpectedresults imply that during β-oxidation, enoyl-CoA hydratase isstereo-specific and only recognizes the ‘S’ isomer. Further,stereoisomeric purity is important for targeting the MFA to hearttissue.

TABLE 5 Biodistribution of isomers and racemic mixture of3-methylheptadecanoic acid and at 5 minutes post-injection OrganS-isomer R-isomer R,S racemic mixture Blood 0.18 ± 0.01 0.30 ± 0.04 0.23± 0.02 Heart 3.44 ± 0.54 1.40 ± 0.23 2.20 ± 0.95 Left Ventricle 4.05 ±0.77 1.50 ± 0.50 2.87 ± 1.01 Right Ventricle 3.40 ± 0.82 0.94 ± 0.462.05 ± 0.54 Heart-to-blood 19.00 4.66  9.56 Left ventricle-to-blood22.00 5.00 12.48 Right ventricle-to- 18.88 3.13  8.90 blood

TABLE 6 Biodistribution of isomers and racemic mixture of3-methylheptadecanoic acid and at 30 minutes post-injection OrganS-isomer R-isomer R,S racemic mixture Blood 0.21 ± 0.04 0.23 ± 0.03 0.20± 0.02 Heart 3.08 ± 0.30 1.07 ± 0.16 2.10 ± 0.41 Left Ventricle 3.81 ±0.37 1.33 ± 0.25 2.81 ± 0.35 Right Ventricle 2.82 ± 0.46 0.93 ± 0.352.10 ± 0.41 Heart-to-blood 14.66 5.95 10.10 Left ventricle-to-blood18.14 5.78 12.48 Right ventricle-to- 18.88 4.00 10.05 blood

Example 4 Imaging Studies in Monkeys with [¹⁸F]-labeled3,4-cyclopropyl-heptadecanoic acid [¹⁸F]-FCPHA

Imaging of pig and monkey hearts after injection of [¹⁸F]FCPHA showed aninitial spike of activity corresponding to blood flow followed by aplateau after 10 minutes (FIG. 3). FIG. 4 represents heart images of apig at 2-8 minutes post-injection of [¹³N]ammonia (18 mCi), and FIG. 5displays heart images of the same pig at 2-8 minutes after intravenousadministration of [¹⁸F]FCPHA (19 mCi). In FIG. 6, the images are acomparison of [¹³N]ammonia (right) and [¹⁸F]FCPHA (left) at 2-8 minutesin a pig. Pig images had a clearer delineation of the heart muscle with[¹⁸F]FCPHA compared with that from [¹³N]ammonia. The [¹⁸F]FCPHA lateimages at 60 minutes exhibited high myocardial activity retention.

The biodistribution of [¹⁸F]-FCPHA was also studied in monkeys. Sixtyminutes after intravenous injection of 5 mCi of [¹⁸F]-FCPHA, theradiolabeled FA analog was visualized by positron emission tomography(PET) and clearly demonstrates myocardial trapping in both left andright ventricles (FIG. 7). FIG. 8 shows transverse heart level slices ofa monkey 60 minutes after administration of [¹⁸F]FCPHA. Blood activityshowed fast clearance in both pig and monkey.

Example 5 Synthesis of Terminally-Labeled[¹⁸F]-fluorophenyl-3,4-cyclopropyl-heptadecanoic Acid

Based on the aforementioned protocol described in Example 1, synthesisof this molecule can be achieved by using a different starting material(such as 10-phenyl-1-decanol). This starting material,10-phenyl-1-decanol, was reacted with triphenylphosphine and dissolvedin benzene. A solution of carbon tetrabromide in benzene was addedslowly and the mixture was refluxed for 90 minutes. The reaction mixturewas then cooled and filtered, and the residue washed three times withportions of petroleum ether. The residue was evaporated to dryness andthen stirred with petroleum ether and left overnight in a freezer. Thesolution was then filtered, the resultant residue washed twice withpetroleum ether, and the combined solution evaporated to dryness. Thisis predicted to yield an alkyl bromide variant of the starting product,wherein the bromine atom is substituted for the primary alcohol moiety.

The brominated compound was subjected to Grignard synthesis as follows.The brominated compound in ether was injected into refluxed ethercontaining magnesium. Reflux continued for 90 minutes under an argon ornitrogen atmosphere. The reaction mixture was then cooled to roomtemperature, then subsequently injected into a ⁷CO₂ trap and thesolution shaken for 5 minutes. The solution was transferred to aseparatory funnel, washed twice with ether, and combined with 1N HCl.The solution was washed twice with water, dried using Na₂SO₄ andevaporated. The resulting compound was predicted to be17-phenyl-1-heptadecanol. The following steps after the second PCCoxidation will continue as described in Example 1. This synthesis yieldsan analog that contains a terminal phenyl group and a cyclopropyl moietyat the 3,4-carbon position (see FIG. 9).

The ¹⁸F or ¹²³I radiolabel can be added to the terminal phenyl moiety byother methods, such as the Schiemann reaction. For ¹⁸F labeling thebenzene moiety can undergo nitration in nitric acid (HNO₃) and sulfuricacid (H₂SO₄), followed by reduction with Sn and HCl. This yields aphenyl moiety labeled with an amino group (aniline). Incubation withNaNO₂ and HCl will convert the amino group to the diazonium ion. Thediazonium salt is then subjected to fluorination with [¹⁸F]-HBF₄. Thisyields a phenyl group mono-substituted with fluorine. For ¹²³I labeling,the benzene substituent will undergo nitration in HNO₃ and H₂SO₄,yielding an aniline group. NaNO₂ and HCl, followed by iodination withpotassium iodide (KI). The iodinated aryl derivative is radiolabeled byan exchange reaction with radioiodide in an acid media at hightemperatures. Other methods include preparation of a correspondingtributyl-tin derivative, followed by electrophilic aromaticradioiodination.

Example 6 Synthesis of endo-[¹⁸F]Fluoro- and [¹²³IIodo-3,4-cyclopropylheptadecanoic Acid Variants

Syntheses of endo-vinylic radiolabeled variants of FCPHA were carriedout essentially as described in Example 1, but with modifications. Asolution of 6-benzyloxy-1-hexanol was treated with PCC in methylenechloride at 25° C. The black mixture was stirred for 2 h and filteredthrough silica gel. The solvent was removed and the crude oil wasseparated on silica gel using hexane/ethyl acetate (90:10).

The resulting aldehyde, 6-benzyloxy-1-hexanal, was mixed with(carbethoxymethylene)triphenylphosphorane in methylene chloride at 25°C. The mixture was stirred overnight, after which the solvent wasremoved and replaced with hexane. Solid matter was removed byfiltration. Chromatography on silica gel, hexane/ethyl acetate (90:10)yielded the corresponding ethyl ester.

Diisobutylaluminum hydride in hexane was added dropwise to the ethylester in methylene chloride at −78° C. The mixture was allowed to slowlywarm to 0° C. and ethyl acetate was added dropwise, followed by ice. Theresultant slurry was acidified with 10% HCl and extracted with ether.The combined ether extracts were washed with brine, dried, and solventwas removed. Chromatography of the crude oil using hexane/ethyl acetate(80:20) yielded the unsaturated primary alcohol.

The unsaturated primary alcohol was subjected to cyclopropanationessentially as described in Charette and coworkers, as well asExample 1. Dry methylene chloride was cooled to −25° C. and placed undera nitrogen atmosphere. Diethyl zinc, followed by 1,2-dimethoxyethane(DME), was added. Diiodomethane was added dropwise to the solution,while maintaining the reaction temperature between −25° C. and −10° C.This solution was then added by double-ended needle, to a solutioncontaining the unsaturated primary alcohol, dioxaborolane (made from(+)N,N,N′,N′-tetramethyl-L-tartaramide and butylboronic acid), and 4 Åmolecular sieves in methylene chloride under nitrogen at −40° C. and−30° C. The reaction mixture was stirred for 2 hours at −25° C., thenallowed to warm to 0° C. Saturated ammonium chloride was added to themixture, then stirred at RT for 16 hours. Solid matter was removed byfiltration and the layers were separated. The aqueous layer wasextracted with methylene chloride, while organic layers were pooled,washed with water, and dried. The crude oil was separated by silica gelchromatography in hexane:ethyl acetate (90:10), yielding the cyclopropylalcohol. This reaction was repeated twice.

Iodine was added in portions to a solution containing the cyclopropylalcohol and triphenylphosphine in dimethylformamide. The dark redmixture was combined with 10% sodium thiosulfate and extracted withether. The combined extract was washed with water, brine, and thesolvent was subsequently removed. Silica gel chromatography inhexane:ethyl acetate (95:5) yielded the alkyl iodide. This alkyl iodide,was combined with sodium cyanide in dimethyl sulfoxide (DMSO), washeated to 80° C. for 2 hours. The mixture was poured into water, and wasthen followed by ether extraction. The combined extract was washed withwater, dried, and solvent removed. The crude oil was separated by silicagel chromatography in hexane:ethyl acetate (80:20) and yielded thecorresponding nitrile compound.

The corresponding nitrile compound was mixed with KOH and water inethylene glycol, then heated for 6 h at 170° C. Once cooled, the mixturewas diluted with 10% HCl and extracted with ether. The combined extractwas dried and the solvent removed. The crude carboxylic acid was treatedwith diazomethane (made from N-methyl-N′-nitro-N-nitrosoguanidine and40% KOH in ether). The reaction mixture was stirred for 1 h beforesolvent removal. Chromatography of the crude oil on silica gel andhexane/ethyl acetate (80:20) yielded the corresponding methyl ester.

The methyl ester was hydrogenated using lithium aluminum hydride, onceagain yielding a primary alcohol. The primary alcohol was substituted byaddition of an alcohol-protecting group, tetrahydropyran (THP).Subsequent hydrogenation of the primary alcohol with 10% palladium oncharcoal (Degussa) in methanol, followed by treatment withN-bromosuccinimide, yielded an alkyl bromide variant, wherein thebromine group was appended on the opposite end from the THP protectinggroup. The alkyl bromide variant was then subjected to treatment withn-butyllithium in hexane, in the presence of an alkyne, which appendedthe alkyne group opposite from the THP moiety. The correspondingcyclopropyl alkyne was hydrogenated with tributyl-tin hydride andiodinated with I₂ to yield an endo-vinyl variant.

The endo-vinyl variant molecule was treated with TosH and CrO₃ underacidic conditions to facilitate removal of the THP protecting group andsubsequent oxidation to the carboxylic acid. The correspondingcarboxylic acid was then subjected to substitution of the iodide withtributyl-tin hydride. Radiolabeled sodium iodide (Na¹²³D) affordedsubstitution of the tributyl-tin moiety with ¹²³I, resulting inendo-[¹²³I]-iodo-3,4-cyclopropylheptadecanoic acid. Alternatively, thetributyl-tin substituted carboxylic acid was treated with ¹⁸F₂ to yieldendo-[¹⁸F]-fluoro-3,4-cyclopropylheptadecanoic acid. A schematicoverview of the synthesis described above is provided in FIG. 10.

Example 7 Synthesis of exo-[¹⁸F]fluoro- and [¹²³Iiodo-3,4-cyclopropylheptadecanoic Acid Variants

An overview of the partial synthesis of the exo-vinyl compounds of3,4-cyclopropyl-heptadecanoic acid is shown in FIG. 12. Synthesis ofradiolabeled exo-vinyl variants of 3,4-cyclopropyl-heptadecanoic acidwas achieved essentially as described in Example 1, up until formationof 9-hydroxy-3,4-cyclopropylheptadecanoate methyl ester. This methylester was oxidized to its corresponding ketone with pyridiniumdichromate (PDC) in methylene chloride at 25° C. The correspondingketone can then be subjected to two different routes, depending on theradiolabeled halogen used.

Vinylic Iodination: Scheme 1

The following syntheses are based on the Homer-Witting reaction, withmodifications described in Stork and Zhao, 1989. Tetrahedron Lett. 30:2173-2174; and McCarthy et al. 1991. J. Am. Chem. Soc. 113: 7439-7440(see also FIG. 12). Pre-formed iodomethyl-triphenylphosphonium iodide,consisting of triphenylphosphine and the iodinated ylide, was addedslowly to a 1 M solution of sodium hexamethyldisilazane intetrahydrofuran (THF). After stirring for one minute, the solution wascooled to −60° C. and hexamethylphosphorictriamide (HMPA) was added,followed by cooling to −78° C. The ketone compound described above wasadded, removed from the cold bath, and stirred for 30 minutes. Hexanewas added to the mixture, followed by column chromatography and NMRanalysis. This afforded substitution of the carbonyl group with theiodinated vinyl group. Subsequent treatment with tributyl-tin hydrideand palladium on carbon yielded substitution of the vinylic iodine withtributyl-tin and conversion of the primary ester group to carboxylicacid. Treatment with radioiodinated sodium iodide (Na¹²³I) resulted inexo-[¹²³I]-iodo-3,4-cyclopropylheptadecanoic acid.

Vinylic Iodination: Scheme 2

Likewise, the addition of the radiolabeled iodine can be directlyachieved, thereby eliminating the intervening vinylic tributyl-tinprecursor molecule (FIG. 12). After substitution of the carbonyl groupwith the iodinated vinyl group, the molecule can then be reacted withcopper and Na¹²³I directly, resulting inexo-[¹²³I]-iodo-3,4-cyclopropyl-heptadecanoic acid.

Vinylic Fluorination: Scheme 1

The vinylic tributyl-tin fatty acid derivative described in Scheme 1 ofvinylic iodination can also be used to directly fluorinate the modifiedfatty acid by substitution of ¹⁸F₂ at the vinylic tributyl-tin moiety(FIG. 12). This yields exo-[¹⁸F]fluoro-3,4-cyclopropyl-heptadecanoicacid.

Vinylic Fluorination: Scheme 2

An alternate synthesis of the vinylic fluorinated variant of3,4-cyclopropyl-heptadecanoic acid can be achieved from the ketonecompound described above (FIG. 12). A modified version of the mechanismis from Burton and Greenlimb 1975. J. Org. Chem. 40: 2796-2801; and inSchlosser and Zimmermann 1969. Synthesis 1: 75-76.Fluoromethyltriphenylphosphonium iodide was synthesized in around-bottomed flask fitted with a water-cooled reflux condenser. Theflask was charged with triphenylphosphine and fluoroiodomethane in drybenzene. The resulting solution was refluxed for 64 hr with moderatestirring. Upon completion of the reaction, the white insolublephosphonium salt was collected on a glass funnel, washed with hotbenzene, and dried in a vacuum dessicator for 12 hr.

A separate flask was equipped with a constant-pressure addition funnelfitted with a nitrogen inlet, and a water-cooled reflux condenser toppedwith a T joint leading to a mineral oil bubbler. The apparatus wasflushed with nitrogen, flame-dried and allowed to cool to roomtemperature while de-aeration with nitrogen was continued. The flask wascharged with fluoromethyltriphenylphosphonium iodide, dry THF, andtoluene. The nitrogen atmosphere was maintained throughout the system.The slurry of phosphonium salt and solvent was moderately stirred whilecooled in a dry ice-isopropanol slush bath. During the cooling process,the addition funnel was charged with n-butyllithium in hexane. Anyappreciable amount of hydrolysis of the organolithium reagent wasavoided by a constant sweep of nitrogen over the addition funnel whenthe stopper was removed and by transferring the base via pipette under adry nitrogen atmosphere. The base was added dropwise over a 23-minuteperiod. After addition, the reaction mixture was stirred for 25 minutesat −78° C. At the end of the 25-minute period, the reaction mixture waswithdrawn under a nitrogen atmosphere and added to ethylene bromide withthe evolution of heat. The reaction was allowed to progress for another25 minutes at −78° C.

The corresponding ketone obtained from PDC oxidation of the methyl ester9-hydroxy-3,4-cyclopropylheptadecanoate was then added. The reactionbetween the ylide and the ketone proceeded for 2 h at −78° C., thenallowed to warm to room temperature, where it was incubated for anadditional 1.5 h. The reaction mixture was then cooled on ice. Potassiumtert-butoxide was added and the mixture was stirred for 2 h at 0° C. Thereaction mixture was centrifuged and decanted, and the precipitatewashed with small portions of THF. The decantates were combined andwashed with saturated aqueous sodium chloride until the aqueous layerwas of neutral pH. The solvent was removed, the dried and filteredorganic material was flash-distilled at reduced pressure, and thensubjected to chromatography and NMR analysis. Base hydrolysis of the¹⁸F-substituted ester resulted in conversion to the correspondingcarboxylic acid. This yieldedexo-[¹⁸F]-fluoro-3,4-cyclopropylheptadecanoic acid.

Example 8 Synthesis of 4 or 5-Membered Ring Substituent Variations ofHeptadecanoic Acid

Variations on heptadecanoic acid have also been synthesized, wherein thecyclopropyl substituent is replaced instead with cyclobutyl orcyclopentyl moieties. Synthesis of these compounds containing variationsof the modified fatty acids described in Examples 1, 4, 5, and 6 areprovided below.

Synthesis of MFAs with the cyclopropyl moiety was described in priorexamples. However, instead of 6-benzyloxy-1-hexanol, which was used asstarting material for many of the aforementioned MFAs, cyclic primaryalcohols (cyclobutanol or cyclopentanol) can be substituted with aprotective group like THP (tetrahydropyran). The mono-protected cyclicalcohol is then subjected to triphenylphosphine in the presence of I₂,which resulted in an alkyl iodide variant, protected at the other end byTHP. Addition of a benzyloxy-alkyne of varying chain length was achievedby addition of n-butyllithium. Hydrogenation in 10% palladium on carbonresulted in a cyclobutyl or cyclopentyl-1-nonanol or heptanol, stillprotected at one end by THP. The chain length of the cyclicmonoprotected primary alcohol depends on the length of thebenzyloxy-alkyne used. These cyclic mono-protected alcohols were used insubsequent reactions, described below. FIG. 14 contains a diagram of thepartial synthesis of these cyclic mono-protected alcohols.

[¹⁸F]-9-fluoro-3,4-cyclobutyl- or[¹⁸F]-9-fluoro-3,4-cyclopentyl-heptadecanoic acid

The cyclic mono-protected nonanol was subjected to PCC oxidation,yielding the corresponding aldehyde. Under a nitrogen atmosphere, octylbromide in dry ether was added to magnesium metal in ether at a rate asto maintain gentle reflux. After addition was complete, the reactionmixture was stirred for 1 h and the cyclic mono-protected nonanal inether, was added dropwise to the reaction mixture at room temperature.The mixture was stirred for 4 h, then poured over ice water, acidifiedin 10% HCl, and extracted with ether. The combined extracts were washedwth brine, dried, and solvent removed. Chromatography on silica gelusing hexane/ethyl acetate (80:20) yielded the cyclic mono-protectedheptadecanol.

The cyclic mono-protected heptadecanol was then subjected tode-protection by treatment with TosH and CrO₃. This yielded theformation of the corresponding cyclic carboxylic acid. The cycliccarboxylic acid was combined with methane sulfonyl chloride and4-dimethyl aminopyridine (DMAP) in methylene chloride/pyridine (90:10).The mixture was stirred for 2 h, then washed with 10% HCl until theaqueous layer was acidic and then washed with 10% NaHCO₃. The crude oilwas chromatographed on silica gel in methylene chloride/methanol (95:5).

¹⁸Fluorine in water was added to a vial containing Kryptofix-222 andK₂CO₃. Water was removed using a nitrogen stream at 115° C. followed byaddition of acetonitrile. To this vial was the mesylated carboxylic acidin acetonitrile. The reaction was heated at 120° C. for 10 minutes,which gave the cyclobutyl or cyclopentyl variant of heptadecanoic acid[¹⁸F]-labeled at the 9-carbon position, followed by silica Sep-Pakpurification in hexane/ethyl acetate (85:15).

Terminally-Labeled [¹⁸F]-fluorophenyl-3,4-cyclobutyl- orcyclopentyl-heptadecanoic acid

Based on the aforementioned protocol described in Example 4, synthesisof this molecule can be achieved by using a different starting material(such as 10-phenyl-1-decanol). This starting material,10-phenyl-1-decanol, was reacted with triphenylphosphine and dissolvedin benzene. A solution of carbon tetrabromide in benzene was addedslowly and the mixture was refluxed for 90 minutes. The reaction mixturewas then cooled and filtered, and the residue washed three times withportions of petroleum ether. The residue was evaporated to dryness andthen stirred with petroleum ether and left overnight in a freezer. Thesolution was then filtered, the resultant residue washed twice withpetroleum ether, and the combined solution evaporated to dryness. Thisis predicted to yield an alkyl bromide variant of the starting product,wherein the bromine atom is substituted for the primary alcohol moiety.

The brominated compound was subjected to Grignard synthesis as follows.The brominated compound in ether was injected into refluxed ethercontaining magnesium. Reflux continued for 90 minutes under an argon ornitrogen atmosphere. The reaction mixture was then cooled to roomtemperature, then subsequently injected into a trap containing acyclobutyl- or cyclopentyl mono-protected 1-heptanol and the solutionshaken for 5 minutes. The solution was transferred to a separatoryfunnel, washed twice with ether, and combined with 1N HCl. The solutionwas washed twice with water, dried using Na₂SO₄ and evaporated. Theresulting heptadecanol compound was predicted to contain a terminalphenyl group at one end, with a cyclobutyl or cyclopentyl substituentfollowed by the THP protective group.

The cyclic mono-protected heptadecanol was then subjected tode-protection by treatment with TosH and CrO₃. This yielded theformation of the corresponding cyclic carboxylic acid. The cycliccarboxylic acid was combined with methane sulfonyl chloride and4-dimethyl aminopyridine (DMAP) in methylene chloride/pyridine (90:10).The mixture was stirred for 2 h, then washed with 10% HCl until theaqueous layer was acidic and then washed with 10% NaHCO₃. The crude oilwas chromatographed on silica gel in methylene chloride/methanol (95:5).

The ¹⁸F or ¹²³I radiolabel can be added to the terminal phenyl moiety byother methods, such as the Schiemann reaction. For ¹⁸F labeling thebenzene moiety can undergo nitration in nitric acid (HNO₃) and sulfuricacid (H₂SO₄), followed by reduction with Sn and HCl. This yields aphenyl moiety labeled with an amino group (aniline). Incubation withNaNO₂ and HCl will convert the amino group to the diazonium ion. Thediazonium salt is then subjected to fluorination with [¹⁸F]-HBF₄. Thisyields a phenyl group mono-substituted with fluorine. For ¹²³I labeling,the benzene substituent will undergo nitration in HNO₃ and H₂SO₄,yielding an aniline group. NaNO₂ and HCl, followed by iodination withpotassium iodide (KI). The iodinated aryl derivative is radiolabeled byan exchange reaction with radioiodide in an acid media at hightemperatures. Other methods include preparation of a correspondingtributyl-tin derivative, followed by electrophilic aromaticradioiodination.

Synthesis of Endo-Vinylic Variants of Cyclobutyl orCyclopentyl-Substituted Heptadecanoic Acid

Cyclobutyl and cyclopentyl variants of the endo-vinylic modified fattyacids described in Example 5 are synthesized essentially as described,however the starting material, a cyclobutyl- or cyclopentyl THPmono-protected heptanol, was treated with N-bromosuccinimide. Thisyielded an alkyl bromide variant, wherein the bromine group was appendedon the opposite end from the THP protecting group. The alkyl bromidevariant was then subjected to treatment with n-butyllithium in hexane,in the presence of an alkyne, which appended the alkyne group oppositefrom the THP moiety. The corresponding cyclopropyl alkyne washydrogenated with tributyl-tin hydride and iodinated with I₂ to yield anendo-vinyl variant.

The endo-vinyl variant molecule was treated with TosH and CrO₃ underacidic conditions to facilitate removal of the THP protecting group andsubsequent oxidation to the carboxylic acid. The correspondingcarboxylic acid was then subjected to substitution of the iodide withtributyl-tin hydride. Radiolabeled sodium iodide (Na¹²³I) affordedsubstitution of the tributyl-tin moiety with ¹²³I, resulting inendo-[¹²³I]-iodo-3,4-cyclobutyl- or cyclopentyl-heptadecanoic acid.Alternatively, the tributyl-tin substituted carboxylic acid was treatedwith ¹⁸F₂ to yield endo-[¹⁸F]-fluoro-3,4-cyclobutyl- orcyclopentyl-heptadecanoic acid.

Synthesis of Exo-Vinylic Variants of Cyclobutyl orCyclopentyl-Substituted Heptadecanoic Acid

The cyclic mono-protected nonanol was subjected to PCC oxidation,yielding the corresponding aldehyde. Under a nitrogen atmosphere, octylbromide in dry ether was added to magnesium metal in ether at a rate asto maintain gentle reflux. After addition was complete, the reactionmixture was stirred for 1 h and the cyclic mono-protected nonanal inether, was added dropwise to the reaction mixture at room temperature.The mixture was stirred for 4 h, then poured over ice water, acidifiedin 10% HCl, and extracted with ether. The combined extracts were washedwth brine, dried, and solvent removed. Chromatography on silica gelusing hexane/ethyl acetate (80:20) yielded the cyclic mono-protectedheptadecanol.

The cyclic mono-protected heptadecanol was then subjected tode-protection by treatment with TosH and CrO₃. This yielded theformation of the corresponding cyclic carboxylic acid. This cycliccarboxylic acid derivative was subjected to vinylic fluorination oriodination as described in Example 6 and modified from Burton andGreenlimb 1975 J. Org. Chem. 40: 2796-2801; and also from Schlösser andZimmermann 1969 Synthesis 1: 75-76.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be apparent to those skilled in the art thatcertain changes and modifications can be practiced. Therefore, thedescription and examples should not be construed as limiting the scopeof the invention, which is delineated by the appended claims.

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1. A radioactively labeled compound that can be taken up by mammaliantissue, wherein the compound is selected from the group consisting of:[¹⁸F]-9-fluoro-3,4-cyclopropylheptadecanoic acid;[¹⁸F]-9-fluoro-3,4-cyclobutylheptadecanoic acid;[¹⁸F]-9-fluoro-3,4-cyclopentylheptadecanoic acid;[¹⁸F]-17-(4-fluorophenyl)-3,4-cyclopropyl-heptadecanoic acid; and[¹⁸F]-17-(4-fluorophenyl)-3,4-cyclobutyl-heptadecanoic acid.
 2. Theradioactively labeled compound of claim 1, wherein said tissue is hearttissue.
 3. The radioactively labeled compound of claim 1, wherein saidcompound emits detectable positrons after being taken up by said tissue.4. The compound [¹⁸F]-9-fluoro-3,4-cyclopropylheptadecanoic acid.
 5. Apharmaceutical composition comprising a radioactively labeled compoundof claim 1 and a pharmaceutically acceptable carrier.
 6. Thepharmaceutical composition of claim 5, wherein the pharmaceuticallyacceptable carrier comprises salts or electrolytes.
 7. Thepharmaceutical composition of claim 5, wherein the pharmaceuticallyacceptable carrier comprises saline.
 8. A pharmaceutical compositioncomprising [¹⁸F]-9-fluoro-3,4-cyclopropylheptadecanoic acid and apharmaceutically acceptable carrier.
 9. The pharmaceutical compositionof claim 8, wherein the pharmaceutically acceptable carrier comprisessalts or electrolytes.
 10. The pharmaceutical composition of claim 8,wherein the pharmaceutically acceptable carrier comprises saline.